Related LOs Sonication gt Prior Viewing ndash IDD-1 Extraction of bacterial protein IDD-6 Extraction of serum protein gt Future Viewing ndash IDD-11 Protein quantification IDD-14 Isoelectric focusing IDD-17 SDS-PAGE

Course Name Sonication on bacteria and serum protein extract Level(UGPG) UG Author(s) Dinesh Raghu Vinayak PachapurMentor Dr Sanjeeva Srivastava

Effect of Sonication on bacterial and Serum protein

extractionSonication is the process by which the high energy sound waves

causing cell lyses rupture of cell wall causes release of contents from the cell The method is more effective in cell disruption Due to

its high efficiency the method is highly suitable fro the protein extraction from the cell and the serum samples

Learning objectivesThe study helps the listener to learn 1Define the mechanism of cell lysing by

sonication2Operate the steps involved in the extraction

technique3Outline the precautions to be followed during the

sonication4Infer the steps involved to perform experiment5Assess the troubleshooting steps involved in the

experiments

5

3

2

4

1

Master Layout

5

3

2

4

1 Serum Sample processing

(Slide4)

Buffer Treatment (Slide 5-6)

Sonication (Slide 7-10)

Bacteria Sample processing (Slide11-12 )

Buffer Treatment (Slide 13)

Sonication (Slide 14-17)

Step 1 T1Sample processing

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Animate removal of sample by opening the freezer and take the tube labeled as sample from -80 C and placing it on the ice for 15min animate the effect with user interaction During 15min zoom the tubes and animate the frozen solution changing to liquid phase Show the user taking the pipette out removal of required amount of serum and transfer it to the new tube Place the old tube in freezer This should happen with user click Kindly redraw the figures

Remove the serum from -80 C and allow it to thaw by keeping it on ice for 15 minTransfer the required amount of the serum to the fresh clean eppendorf tube for further sample processing Transfer of sample must be a quick process

Step 2

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Show phosphate buffer bottle instruct user to set the pipette to 500ul pipette out the buffer as the user clicks on it and adding it to the tube labeled as serum for dilution The user should click on the pipette for the action to be done Kindly redraw the figures

Dilute the serum 5 times using phosphate buffer of pH 74 which helps to maintain the buffer during the sonication step

T2Buffer addition

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Instruct user to vortex the tube hand animate by picking the tube and placing on top of rubber pad for vortex and clicking ldquoONrdquo button During vortex animate the mixing of solution The user should click on start button for vortex kindly redraw the figures

Vortex the sample to achieve uniform mixingVortex the sample to achieve

uniform mixing

T2Buffer addition

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touching the solution in the tube animate like the user adjusting the rod and dipping the tube in ice Now display the screen of sonicator to make the necessary setup like pulses time and amplitude with help of user interaction and use the required values from the right hand side to set the parameter settingsuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5sec 20 amplitude with 5sec gap Sonication carries out cell lysis which helps for protein extraction

T3Sonication

Step 3 T3Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves split the aggregated serum proteins and thereby makes the sample easy for further analysis

Zoom in a aggregation as in the figure from the tube Show the sound waves hitting the those aggregation and causing separation splitting of aggreagated protein and the release of contents Please redraw the figure

sonication

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

After sonication zoom the tube instruct user to click on the tube for the contents display Animate the user interaction

After sonication the cells are lysed now serum contains high abundance proteins like albumin IgA IgG haptoglobin along with low abundance protein High abundance protein will interfere in gel separation and prevent the separation of low abundance proteins After sonication steps the sample is taken for further purification process

T3Sonication

Step 3

5

2

1

4

3

Purification steps same as IDD-6 Extraction of serum protein from Slide 17-32 For more information on the extraction steps go through future viewing IDD

Step 1 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Inoculation same as in IDD-1 Extraction of bacterial protein

Instruct user to open the lid of centrifuge and rotor Zoom in the rotor balance equal number of tubes inside the rotor Close the lid of rotor and of centrifuge with hand action Instruct user to set the rpm temperature and time parameters animate this in the display where user can increase and decrease the values of set parameters Animate the clock for 30min Kindly redraw the figures

Transfer the bacterial culture into the clean centrifuge tube under aseptic condition Centrifuge the culture for 10 min at 12000 rpm maintaining at 4rsquoC

Step 2 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

After 10min instruct user to open the lid of centrifuge rotor and animate the hand action to left the tube from rotor Now zoom the tube with pellet on bottom and liquid (supernatant) over it as shown in figure Now pipette out top portion (supernatant) completely into the discard the action should take place only when the user clicks on the pipette and the tube Kindly redraw the figures

Remove the supernatant completely without disturbing the pellet now take the pellet for further processing

Step 2 T2 Buffer treatment

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Show phosphate buffer bottle instruct user to pipette into the tube The user should click on the pipette for the action to be done Kindly redraw the figures

Wash the pellet with phosphate buffer thoroughly to remove the excess broth Once broth is removed completely cell lysis need to be carried out

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touches the solution in the tube Now display the screen of sonicator to make the necessary setup with help of user interactionuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5 sec 20 amplitude with 5 sec gap Sonication carries out cell lysis which helps for protein extraction

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves break open the cell wall and the contents are released into the buffer

High frequency sound waves

Zoom in a cell from the tube Show the sound waves hitting the cell and causing cell lyses and the release of contents Animate cell breaking with release of content into the tube from inside the cell Please redraw the figure

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Carry out the 3 centrifuge process as described earlier The user should click on ldquo Startrdquo for the centrifugation to on Please redraw the figure

Centrifuge the contents to remove the debris and collect the supernatant for further processing

Step 3

5

2

1

4

3

Extraction steps are same as IDD-1 Extraction of bacterial protein from Slide 16-32For more information on the purification steps go through future viewing IDD

Animation areaInteraction 1 slide-14 user carrying out sonication

without cooling on ice bath no gap between the bursts and proceeds further

Instructions let user place the tube on ice packs at the time of sonication and gives a time delay for few seconds between the bursts

Interaction 2 slide-14 user carrying out sonication for longer time

Instructions animate the tube getting hot and starts melting Excessive sonication will lead to heating and ice packs helps down in cooling the sample

Instructions Working area

Credits

Name of the sectionstage BACTERIAL CULTUREInteractivityarea

Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Button 01

Button 02

Button 03

Tab 01

Slide 4 Slide 7-10

Slide 5-6

Slide 11-12

Slide 14-17

Slide 13

QuestionnaireAPPENDIX

1

Question 1Amplitude used for the sonication of serum isa)20b)30c)40d)50

Answer 20

What is the purpose of sonication in bacterial protein extraction

a) To mix the cultureb) To denature the proteinc) To denature the

nucleotidesd) To lyse the cellsAnswer To lyse the cells

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

Learning objectivesThe study helps the listener to learn 1Define the mechanism of cell lysing by

sonication2Operate the steps involved in the extraction

technique3Outline the precautions to be followed during the

sonication4Infer the steps involved to perform experiment5Assess the troubleshooting steps involved in the

experiments

5

3

2

4

1

Master Layout

5

3

2

4

1 Serum Sample processing

(Slide4)

Buffer Treatment (Slide 5-6)

Sonication (Slide 7-10)

Bacteria Sample processing (Slide11-12 )

Buffer Treatment (Slide 13)

Sonication (Slide 14-17)

Step 1 T1Sample processing

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Animate removal of sample by opening the freezer and take the tube labeled as sample from -80 C and placing it on the ice for 15min animate the effect with user interaction During 15min zoom the tubes and animate the frozen solution changing to liquid phase Show the user taking the pipette out removal of required amount of serum and transfer it to the new tube Place the old tube in freezer This should happen with user click Kindly redraw the figures

Remove the serum from -80 C and allow it to thaw by keeping it on ice for 15 minTransfer the required amount of the serum to the fresh clean eppendorf tube for further sample processing Transfer of sample must be a quick process

Step 2

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Show phosphate buffer bottle instruct user to set the pipette to 500ul pipette out the buffer as the user clicks on it and adding it to the tube labeled as serum for dilution The user should click on the pipette for the action to be done Kindly redraw the figures

Dilute the serum 5 times using phosphate buffer of pH 74 which helps to maintain the buffer during the sonication step

T2Buffer addition

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Instruct user to vortex the tube hand animate by picking the tube and placing on top of rubber pad for vortex and clicking ldquoONrdquo button During vortex animate the mixing of solution The user should click on start button for vortex kindly redraw the figures

Vortex the sample to achieve uniform mixingVortex the sample to achieve

uniform mixing

T2Buffer addition

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touching the solution in the tube animate like the user adjusting the rod and dipping the tube in ice Now display the screen of sonicator to make the necessary setup like pulses time and amplitude with help of user interaction and use the required values from the right hand side to set the parameter settingsuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5sec 20 amplitude with 5sec gap Sonication carries out cell lysis which helps for protein extraction

T3Sonication

Step 3 T3Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves split the aggregated serum proteins and thereby makes the sample easy for further analysis

Zoom in a aggregation as in the figure from the tube Show the sound waves hitting the those aggregation and causing separation splitting of aggreagated protein and the release of contents Please redraw the figure

sonication

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

After sonication zoom the tube instruct user to click on the tube for the contents display Animate the user interaction

After sonication the cells are lysed now serum contains high abundance proteins like albumin IgA IgG haptoglobin along with low abundance protein High abundance protein will interfere in gel separation and prevent the separation of low abundance proteins After sonication steps the sample is taken for further purification process

T3Sonication

Step 3

5

2

1

4

3

Purification steps same as IDD-6 Extraction of serum protein from Slide 17-32 For more information on the extraction steps go through future viewing IDD

Step 1 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Inoculation same as in IDD-1 Extraction of bacterial protein

Instruct user to open the lid of centrifuge and rotor Zoom in the rotor balance equal number of tubes inside the rotor Close the lid of rotor and of centrifuge with hand action Instruct user to set the rpm temperature and time parameters animate this in the display where user can increase and decrease the values of set parameters Animate the clock for 30min Kindly redraw the figures

Transfer the bacterial culture into the clean centrifuge tube under aseptic condition Centrifuge the culture for 10 min at 12000 rpm maintaining at 4rsquoC

Step 2 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

After 10min instruct user to open the lid of centrifuge rotor and animate the hand action to left the tube from rotor Now zoom the tube with pellet on bottom and liquid (supernatant) over it as shown in figure Now pipette out top portion (supernatant) completely into the discard the action should take place only when the user clicks on the pipette and the tube Kindly redraw the figures

Remove the supernatant completely without disturbing the pellet now take the pellet for further processing

Step 2 T2 Buffer treatment

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Show phosphate buffer bottle instruct user to pipette into the tube The user should click on the pipette for the action to be done Kindly redraw the figures

Wash the pellet with phosphate buffer thoroughly to remove the excess broth Once broth is removed completely cell lysis need to be carried out

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touches the solution in the tube Now display the screen of sonicator to make the necessary setup with help of user interactionuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5 sec 20 amplitude with 5 sec gap Sonication carries out cell lysis which helps for protein extraction

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves break open the cell wall and the contents are released into the buffer

High frequency sound waves

Zoom in a cell from the tube Show the sound waves hitting the cell and causing cell lyses and the release of contents Animate cell breaking with release of content into the tube from inside the cell Please redraw the figure

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Carry out the 3 centrifuge process as described earlier The user should click on ldquo Startrdquo for the centrifugation to on Please redraw the figure

Centrifuge the contents to remove the debris and collect the supernatant for further processing

Step 3

5

2

1

4

3

Extraction steps are same as IDD-1 Extraction of bacterial protein from Slide 16-32For more information on the purification steps go through future viewing IDD

Animation areaInteraction 1 slide-14 user carrying out sonication

without cooling on ice bath no gap between the bursts and proceeds further

Instructions let user place the tube on ice packs at the time of sonication and gives a time delay for few seconds between the bursts

Interaction 2 slide-14 user carrying out sonication for longer time

Instructions animate the tube getting hot and starts melting Excessive sonication will lead to heating and ice packs helps down in cooling the sample

Instructions Working area

Credits

Name of the sectionstage BACTERIAL CULTUREInteractivityarea

Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Button 01

Button 02

Button 03

Tab 01

Slide 4 Slide 7-10

Slide 5-6

Slide 11-12

Slide 14-17

Slide 13

QuestionnaireAPPENDIX

1

Question 1Amplitude used for the sonication of serum isa)20b)30c)40d)50

Answer 20

What is the purpose of sonication in bacterial protein extraction

a) To mix the cultureb) To denature the proteinc) To denature the

nucleotidesd) To lyse the cellsAnswer To lyse the cells

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

Master Layout

5

3

2

4

1 Serum Sample processing

(Slide4)

Buffer Treatment (Slide 5-6)

Sonication (Slide 7-10)

Bacteria Sample processing (Slide11-12 )

Buffer Treatment (Slide 13)

Sonication (Slide 14-17)

Step 1 T1Sample processing

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Animate removal of sample by opening the freezer and take the tube labeled as sample from -80 C and placing it on the ice for 15min animate the effect with user interaction During 15min zoom the tubes and animate the frozen solution changing to liquid phase Show the user taking the pipette out removal of required amount of serum and transfer it to the new tube Place the old tube in freezer This should happen with user click Kindly redraw the figures

Remove the serum from -80 C and allow it to thaw by keeping it on ice for 15 minTransfer the required amount of the serum to the fresh clean eppendorf tube for further sample processing Transfer of sample must be a quick process

Step 2

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Show phosphate buffer bottle instruct user to set the pipette to 500ul pipette out the buffer as the user clicks on it and adding it to the tube labeled as serum for dilution The user should click on the pipette for the action to be done Kindly redraw the figures

Dilute the serum 5 times using phosphate buffer of pH 74 which helps to maintain the buffer during the sonication step

T2Buffer addition

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Instruct user to vortex the tube hand animate by picking the tube and placing on top of rubber pad for vortex and clicking ldquoONrdquo button During vortex animate the mixing of solution The user should click on start button for vortex kindly redraw the figures

Vortex the sample to achieve uniform mixingVortex the sample to achieve

uniform mixing

T2Buffer addition

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touching the solution in the tube animate like the user adjusting the rod and dipping the tube in ice Now display the screen of sonicator to make the necessary setup like pulses time and amplitude with help of user interaction and use the required values from the right hand side to set the parameter settingsuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5sec 20 amplitude with 5sec gap Sonication carries out cell lysis which helps for protein extraction

T3Sonication

Step 3 T3Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves split the aggregated serum proteins and thereby makes the sample easy for further analysis

Zoom in a aggregation as in the figure from the tube Show the sound waves hitting the those aggregation and causing separation splitting of aggreagated protein and the release of contents Please redraw the figure

sonication

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

After sonication zoom the tube instruct user to click on the tube for the contents display Animate the user interaction

After sonication the cells are lysed now serum contains high abundance proteins like albumin IgA IgG haptoglobin along with low abundance protein High abundance protein will interfere in gel separation and prevent the separation of low abundance proteins After sonication steps the sample is taken for further purification process

T3Sonication

Step 3

5

2

1

4

3

Purification steps same as IDD-6 Extraction of serum protein from Slide 17-32 For more information on the extraction steps go through future viewing IDD

Step 1 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Inoculation same as in IDD-1 Extraction of bacterial protein

Instruct user to open the lid of centrifuge and rotor Zoom in the rotor balance equal number of tubes inside the rotor Close the lid of rotor and of centrifuge with hand action Instruct user to set the rpm temperature and time parameters animate this in the display where user can increase and decrease the values of set parameters Animate the clock for 30min Kindly redraw the figures

Transfer the bacterial culture into the clean centrifuge tube under aseptic condition Centrifuge the culture for 10 min at 12000 rpm maintaining at 4rsquoC

Step 2 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

After 10min instruct user to open the lid of centrifuge rotor and animate the hand action to left the tube from rotor Now zoom the tube with pellet on bottom and liquid (supernatant) over it as shown in figure Now pipette out top portion (supernatant) completely into the discard the action should take place only when the user clicks on the pipette and the tube Kindly redraw the figures

Remove the supernatant completely without disturbing the pellet now take the pellet for further processing

Step 2 T2 Buffer treatment

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Show phosphate buffer bottle instruct user to pipette into the tube The user should click on the pipette for the action to be done Kindly redraw the figures

Wash the pellet with phosphate buffer thoroughly to remove the excess broth Once broth is removed completely cell lysis need to be carried out

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touches the solution in the tube Now display the screen of sonicator to make the necessary setup with help of user interactionuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5 sec 20 amplitude with 5 sec gap Sonication carries out cell lysis which helps for protein extraction

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves break open the cell wall and the contents are released into the buffer

High frequency sound waves

Zoom in a cell from the tube Show the sound waves hitting the cell and causing cell lyses and the release of contents Animate cell breaking with release of content into the tube from inside the cell Please redraw the figure

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Carry out the 3 centrifuge process as described earlier The user should click on ldquo Startrdquo for the centrifugation to on Please redraw the figure

Centrifuge the contents to remove the debris and collect the supernatant for further processing

Step 3

5

2

1

4

3

Extraction steps are same as IDD-1 Extraction of bacterial protein from Slide 16-32For more information on the purification steps go through future viewing IDD

Animation areaInteraction 1 slide-14 user carrying out sonication

without cooling on ice bath no gap between the bursts and proceeds further

Instructions let user place the tube on ice packs at the time of sonication and gives a time delay for few seconds between the bursts

Interaction 2 slide-14 user carrying out sonication for longer time

Instructions animate the tube getting hot and starts melting Excessive sonication will lead to heating and ice packs helps down in cooling the sample

Instructions Working area

Credits

Name of the sectionstage BACTERIAL CULTUREInteractivityarea

Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Button 01

Button 02

Button 03

Tab 01

Slide 4 Slide 7-10

Slide 5-6

Slide 11-12

Slide 14-17

Slide 13

QuestionnaireAPPENDIX

1

Question 1Amplitude used for the sonication of serum isa)20b)30c)40d)50

Answer 20

What is the purpose of sonication in bacterial protein extraction

a) To mix the cultureb) To denature the proteinc) To denature the

nucleotidesd) To lyse the cellsAnswer To lyse the cells

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

Step 1 T1Sample processing

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Animate removal of sample by opening the freezer and take the tube labeled as sample from -80 C and placing it on the ice for 15min animate the effect with user interaction During 15min zoom the tubes and animate the frozen solution changing to liquid phase Show the user taking the pipette out removal of required amount of serum and transfer it to the new tube Place the old tube in freezer This should happen with user click Kindly redraw the figures

Remove the serum from -80 C and allow it to thaw by keeping it on ice for 15 minTransfer the required amount of the serum to the fresh clean eppendorf tube for further sample processing Transfer of sample must be a quick process

Step 2

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Show phosphate buffer bottle instruct user to set the pipette to 500ul pipette out the buffer as the user clicks on it and adding it to the tube labeled as serum for dilution The user should click on the pipette for the action to be done Kindly redraw the figures

Dilute the serum 5 times using phosphate buffer of pH 74 which helps to maintain the buffer during the sonication step

T2Buffer addition

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Instruct user to vortex the tube hand animate by picking the tube and placing on top of rubber pad for vortex and clicking ldquoONrdquo button During vortex animate the mixing of solution The user should click on start button for vortex kindly redraw the figures

Vortex the sample to achieve uniform mixingVortex the sample to achieve

uniform mixing

T2Buffer addition

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touching the solution in the tube animate like the user adjusting the rod and dipping the tube in ice Now display the screen of sonicator to make the necessary setup like pulses time and amplitude with help of user interaction and use the required values from the right hand side to set the parameter settingsuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5sec 20 amplitude with 5sec gap Sonication carries out cell lysis which helps for protein extraction

T3Sonication

Step 3 T3Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves split the aggregated serum proteins and thereby makes the sample easy for further analysis

Zoom in a aggregation as in the figure from the tube Show the sound waves hitting the those aggregation and causing separation splitting of aggreagated protein and the release of contents Please redraw the figure

sonication

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

After sonication zoom the tube instruct user to click on the tube for the contents display Animate the user interaction

After sonication the cells are lysed now serum contains high abundance proteins like albumin IgA IgG haptoglobin along with low abundance protein High abundance protein will interfere in gel separation and prevent the separation of low abundance proteins After sonication steps the sample is taken for further purification process

T3Sonication

Step 3

5

2

1

4

3

Purification steps same as IDD-6 Extraction of serum protein from Slide 17-32 For more information on the extraction steps go through future viewing IDD

Step 1 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Inoculation same as in IDD-1 Extraction of bacterial protein

Instruct user to open the lid of centrifuge and rotor Zoom in the rotor balance equal number of tubes inside the rotor Close the lid of rotor and of centrifuge with hand action Instruct user to set the rpm temperature and time parameters animate this in the display where user can increase and decrease the values of set parameters Animate the clock for 30min Kindly redraw the figures

Transfer the bacterial culture into the clean centrifuge tube under aseptic condition Centrifuge the culture for 10 min at 12000 rpm maintaining at 4rsquoC

Step 2 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

After 10min instruct user to open the lid of centrifuge rotor and animate the hand action to left the tube from rotor Now zoom the tube with pellet on bottom and liquid (supernatant) over it as shown in figure Now pipette out top portion (supernatant) completely into the discard the action should take place only when the user clicks on the pipette and the tube Kindly redraw the figures

Remove the supernatant completely without disturbing the pellet now take the pellet for further processing

Step 2 T2 Buffer treatment

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Show phosphate buffer bottle instruct user to pipette into the tube The user should click on the pipette for the action to be done Kindly redraw the figures

Wash the pellet with phosphate buffer thoroughly to remove the excess broth Once broth is removed completely cell lysis need to be carried out

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touches the solution in the tube Now display the screen of sonicator to make the necessary setup with help of user interactionuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5 sec 20 amplitude with 5 sec gap Sonication carries out cell lysis which helps for protein extraction

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves break open the cell wall and the contents are released into the buffer

High frequency sound waves

Zoom in a cell from the tube Show the sound waves hitting the cell and causing cell lyses and the release of contents Animate cell breaking with release of content into the tube from inside the cell Please redraw the figure

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Carry out the 3 centrifuge process as described earlier The user should click on ldquo Startrdquo for the centrifugation to on Please redraw the figure

Centrifuge the contents to remove the debris and collect the supernatant for further processing

Step 3

5

2

1

4

3

Extraction steps are same as IDD-1 Extraction of bacterial protein from Slide 16-32For more information on the purification steps go through future viewing IDD

Animation areaInteraction 1 slide-14 user carrying out sonication

without cooling on ice bath no gap between the bursts and proceeds further

Instructions let user place the tube on ice packs at the time of sonication and gives a time delay for few seconds between the bursts

Interaction 2 slide-14 user carrying out sonication for longer time

Instructions animate the tube getting hot and starts melting Excessive sonication will lead to heating and ice packs helps down in cooling the sample

Instructions Working area

Credits

Name of the sectionstage BACTERIAL CULTUREInteractivityarea

Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Button 01

Button 02

Button 03

Tab 01

Slide 4 Slide 7-10

Slide 5-6

Slide 11-12

Slide 14-17

Slide 13

QuestionnaireAPPENDIX

1

Question 1Amplitude used for the sonication of serum isa)20b)30c)40d)50

Answer 20

What is the purpose of sonication in bacterial protein extraction

a) To mix the cultureb) To denature the proteinc) To denature the

nucleotidesd) To lyse the cellsAnswer To lyse the cells

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

Step 2

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Show phosphate buffer bottle instruct user to set the pipette to 500ul pipette out the buffer as the user clicks on it and adding it to the tube labeled as serum for dilution The user should click on the pipette for the action to be done Kindly redraw the figures

Dilute the serum 5 times using phosphate buffer of pH 74 which helps to maintain the buffer during the sonication step

T2Buffer addition

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Instruct user to vortex the tube hand animate by picking the tube and placing on top of rubber pad for vortex and clicking ldquoONrdquo button During vortex animate the mixing of solution The user should click on start button for vortex kindly redraw the figures

Vortex the sample to achieve uniform mixingVortex the sample to achieve

uniform mixing

T2Buffer addition

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touching the solution in the tube animate like the user adjusting the rod and dipping the tube in ice Now display the screen of sonicator to make the necessary setup like pulses time and amplitude with help of user interaction and use the required values from the right hand side to set the parameter settingsuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5sec 20 amplitude with 5sec gap Sonication carries out cell lysis which helps for protein extraction

T3Sonication

Step 3 T3Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves split the aggregated serum proteins and thereby makes the sample easy for further analysis

Zoom in a aggregation as in the figure from the tube Show the sound waves hitting the those aggregation and causing separation splitting of aggreagated protein and the release of contents Please redraw the figure

sonication

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

After sonication zoom the tube instruct user to click on the tube for the contents display Animate the user interaction

After sonication the cells are lysed now serum contains high abundance proteins like albumin IgA IgG haptoglobin along with low abundance protein High abundance protein will interfere in gel separation and prevent the separation of low abundance proteins After sonication steps the sample is taken for further purification process

T3Sonication

Step 3

5

2

1

4

3

Purification steps same as IDD-6 Extraction of serum protein from Slide 17-32 For more information on the extraction steps go through future viewing IDD

Step 1 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Inoculation same as in IDD-1 Extraction of bacterial protein

Instruct user to open the lid of centrifuge and rotor Zoom in the rotor balance equal number of tubes inside the rotor Close the lid of rotor and of centrifuge with hand action Instruct user to set the rpm temperature and time parameters animate this in the display where user can increase and decrease the values of set parameters Animate the clock for 30min Kindly redraw the figures

Transfer the bacterial culture into the clean centrifuge tube under aseptic condition Centrifuge the culture for 10 min at 12000 rpm maintaining at 4rsquoC

Step 2 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

After 10min instruct user to open the lid of centrifuge rotor and animate the hand action to left the tube from rotor Now zoom the tube with pellet on bottom and liquid (supernatant) over it as shown in figure Now pipette out top portion (supernatant) completely into the discard the action should take place only when the user clicks on the pipette and the tube Kindly redraw the figures

Remove the supernatant completely without disturbing the pellet now take the pellet for further processing

Step 2 T2 Buffer treatment

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Show phosphate buffer bottle instruct user to pipette into the tube The user should click on the pipette for the action to be done Kindly redraw the figures

Wash the pellet with phosphate buffer thoroughly to remove the excess broth Once broth is removed completely cell lysis need to be carried out

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touches the solution in the tube Now display the screen of sonicator to make the necessary setup with help of user interactionuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5 sec 20 amplitude with 5 sec gap Sonication carries out cell lysis which helps for protein extraction

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves break open the cell wall and the contents are released into the buffer

High frequency sound waves

Zoom in a cell from the tube Show the sound waves hitting the cell and causing cell lyses and the release of contents Animate cell breaking with release of content into the tube from inside the cell Please redraw the figure

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Carry out the 3 centrifuge process as described earlier The user should click on ldquo Startrdquo for the centrifugation to on Please redraw the figure

Centrifuge the contents to remove the debris and collect the supernatant for further processing

Step 3

5

2

1

4

3

Extraction steps are same as IDD-1 Extraction of bacterial protein from Slide 16-32For more information on the purification steps go through future viewing IDD

Animation areaInteraction 1 slide-14 user carrying out sonication

without cooling on ice bath no gap between the bursts and proceeds further

Instructions let user place the tube on ice packs at the time of sonication and gives a time delay for few seconds between the bursts

Interaction 2 slide-14 user carrying out sonication for longer time

Instructions animate the tube getting hot and starts melting Excessive sonication will lead to heating and ice packs helps down in cooling the sample

Instructions Working area

Credits

Name of the sectionstage BACTERIAL CULTUREInteractivityarea

Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Button 01

Button 02

Button 03

Tab 01

Slide 4 Slide 7-10

Slide 5-6

Slide 11-12

Slide 14-17

Slide 13

QuestionnaireAPPENDIX

1

Question 1Amplitude used for the sonication of serum isa)20b)30c)40d)50

Answer 20

What is the purpose of sonication in bacterial protein extraction

a) To mix the cultureb) To denature the proteinc) To denature the

nucleotidesd) To lyse the cellsAnswer To lyse the cells

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Instruct user to vortex the tube hand animate by picking the tube and placing on top of rubber pad for vortex and clicking ldquoONrdquo button During vortex animate the mixing of solution The user should click on start button for vortex kindly redraw the figures

Vortex the sample to achieve uniform mixingVortex the sample to achieve

uniform mixing

T2Buffer addition

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touching the solution in the tube animate like the user adjusting the rod and dipping the tube in ice Now display the screen of sonicator to make the necessary setup like pulses time and amplitude with help of user interaction and use the required values from the right hand side to set the parameter settingsuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5sec 20 amplitude with 5sec gap Sonication carries out cell lysis which helps for protein extraction

T3Sonication

Step 3 T3Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves split the aggregated serum proteins and thereby makes the sample easy for further analysis

Zoom in a aggregation as in the figure from the tube Show the sound waves hitting the those aggregation and causing separation splitting of aggreagated protein and the release of contents Please redraw the figure

sonication

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

After sonication zoom the tube instruct user to click on the tube for the contents display Animate the user interaction

After sonication the cells are lysed now serum contains high abundance proteins like albumin IgA IgG haptoglobin along with low abundance protein High abundance protein will interfere in gel separation and prevent the separation of low abundance proteins After sonication steps the sample is taken for further purification process

T3Sonication

Step 3

5

2

1

4

3

Purification steps same as IDD-6 Extraction of serum protein from Slide 17-32 For more information on the extraction steps go through future viewing IDD

Step 1 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Inoculation same as in IDD-1 Extraction of bacterial protein

Instruct user to open the lid of centrifuge and rotor Zoom in the rotor balance equal number of tubes inside the rotor Close the lid of rotor and of centrifuge with hand action Instruct user to set the rpm temperature and time parameters animate this in the display where user can increase and decrease the values of set parameters Animate the clock for 30min Kindly redraw the figures

Transfer the bacterial culture into the clean centrifuge tube under aseptic condition Centrifuge the culture for 10 min at 12000 rpm maintaining at 4rsquoC

Step 2 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

After 10min instruct user to open the lid of centrifuge rotor and animate the hand action to left the tube from rotor Now zoom the tube with pellet on bottom and liquid (supernatant) over it as shown in figure Now pipette out top portion (supernatant) completely into the discard the action should take place only when the user clicks on the pipette and the tube Kindly redraw the figures

Remove the supernatant completely without disturbing the pellet now take the pellet for further processing

Step 2 T2 Buffer treatment

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Show phosphate buffer bottle instruct user to pipette into the tube The user should click on the pipette for the action to be done Kindly redraw the figures

Wash the pellet with phosphate buffer thoroughly to remove the excess broth Once broth is removed completely cell lysis need to be carried out

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touches the solution in the tube Now display the screen of sonicator to make the necessary setup with help of user interactionuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5 sec 20 amplitude with 5 sec gap Sonication carries out cell lysis which helps for protein extraction

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves break open the cell wall and the contents are released into the buffer

High frequency sound waves

Zoom in a cell from the tube Show the sound waves hitting the cell and causing cell lyses and the release of contents Animate cell breaking with release of content into the tube from inside the cell Please redraw the figure

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Carry out the 3 centrifuge process as described earlier The user should click on ldquo Startrdquo for the centrifugation to on Please redraw the figure

Centrifuge the contents to remove the debris and collect the supernatant for further processing

Step 3

5

2

1

4

3

Extraction steps are same as IDD-1 Extraction of bacterial protein from Slide 16-32For more information on the purification steps go through future viewing IDD

Animation areaInteraction 1 slide-14 user carrying out sonication

without cooling on ice bath no gap between the bursts and proceeds further

Instructions let user place the tube on ice packs at the time of sonication and gives a time delay for few seconds between the bursts

Interaction 2 slide-14 user carrying out sonication for longer time

Instructions animate the tube getting hot and starts melting Excessive sonication will lead to heating and ice packs helps down in cooling the sample

Instructions Working area

Credits

Name of the sectionstage BACTERIAL CULTUREInteractivityarea

Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Button 01

Button 02

Button 03

Tab 01

Slide 4 Slide 7-10

Slide 5-6

Slide 11-12

Slide 14-17

Slide 13

QuestionnaireAPPENDIX

1

Question 1Amplitude used for the sonication of serum isa)20b)30c)40d)50

Answer 20

What is the purpose of sonication in bacterial protein extraction

a) To mix the cultureb) To denature the proteinc) To denature the

nucleotidesd) To lyse the cellsAnswer To lyse the cells

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touching the solution in the tube animate like the user adjusting the rod and dipping the tube in ice Now display the screen of sonicator to make the necessary setup like pulses time and amplitude with help of user interaction and use the required values from the right hand side to set the parameter settingsuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5sec 20 amplitude with 5sec gap Sonication carries out cell lysis which helps for protein extraction

T3Sonication

Step 3 T3Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves split the aggregated serum proteins and thereby makes the sample easy for further analysis

Zoom in a aggregation as in the figure from the tube Show the sound waves hitting the those aggregation and causing separation splitting of aggreagated protein and the release of contents Please redraw the figure

sonication

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

After sonication zoom the tube instruct user to click on the tube for the contents display Animate the user interaction

After sonication the cells are lysed now serum contains high abundance proteins like albumin IgA IgG haptoglobin along with low abundance protein High abundance protein will interfere in gel separation and prevent the separation of low abundance proteins After sonication steps the sample is taken for further purification process

T3Sonication

Step 3

5

2

1

4

3

Purification steps same as IDD-6 Extraction of serum protein from Slide 17-32 For more information on the extraction steps go through future viewing IDD

Step 1 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Inoculation same as in IDD-1 Extraction of bacterial protein

Instruct user to open the lid of centrifuge and rotor Zoom in the rotor balance equal number of tubes inside the rotor Close the lid of rotor and of centrifuge with hand action Instruct user to set the rpm temperature and time parameters animate this in the display where user can increase and decrease the values of set parameters Animate the clock for 30min Kindly redraw the figures

Transfer the bacterial culture into the clean centrifuge tube under aseptic condition Centrifuge the culture for 10 min at 12000 rpm maintaining at 4rsquoC

Step 2 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

After 10min instruct user to open the lid of centrifuge rotor and animate the hand action to left the tube from rotor Now zoom the tube with pellet on bottom and liquid (supernatant) over it as shown in figure Now pipette out top portion (supernatant) completely into the discard the action should take place only when the user clicks on the pipette and the tube Kindly redraw the figures

Remove the supernatant completely without disturbing the pellet now take the pellet for further processing

Step 2 T2 Buffer treatment

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Show phosphate buffer bottle instruct user to pipette into the tube The user should click on the pipette for the action to be done Kindly redraw the figures

Wash the pellet with phosphate buffer thoroughly to remove the excess broth Once broth is removed completely cell lysis need to be carried out

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touches the solution in the tube Now display the screen of sonicator to make the necessary setup with help of user interactionuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5 sec 20 amplitude with 5 sec gap Sonication carries out cell lysis which helps for protein extraction

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves break open the cell wall and the contents are released into the buffer

High frequency sound waves

Zoom in a cell from the tube Show the sound waves hitting the cell and causing cell lyses and the release of contents Animate cell breaking with release of content into the tube from inside the cell Please redraw the figure

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Carry out the 3 centrifuge process as described earlier The user should click on ldquo Startrdquo for the centrifugation to on Please redraw the figure

Centrifuge the contents to remove the debris and collect the supernatant for further processing

Step 3

5

2

1

4

3

Extraction steps are same as IDD-1 Extraction of bacterial protein from Slide 16-32For more information on the purification steps go through future viewing IDD

Animation areaInteraction 1 slide-14 user carrying out sonication

without cooling on ice bath no gap between the bursts and proceeds further

Instructions let user place the tube on ice packs at the time of sonication and gives a time delay for few seconds between the bursts

Interaction 2 slide-14 user carrying out sonication for longer time

Instructions animate the tube getting hot and starts melting Excessive sonication will lead to heating and ice packs helps down in cooling the sample

Instructions Working area

Credits

Name of the sectionstage BACTERIAL CULTUREInteractivityarea

Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Button 01

Button 02

Button 03

Tab 01

Slide 4 Slide 7-10

Slide 5-6

Slide 11-12

Slide 14-17

Slide 13

QuestionnaireAPPENDIX

1

Question 1Amplitude used for the sonication of serum isa)20b)30c)40d)50

Answer 20

What is the purpose of sonication in bacterial protein extraction

a) To mix the cultureb) To denature the proteinc) To denature the

nucleotidesd) To lyse the cellsAnswer To lyse the cells

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

Step 3 T3Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves split the aggregated serum proteins and thereby makes the sample easy for further analysis

Zoom in a aggregation as in the figure from the tube Show the sound waves hitting the those aggregation and causing separation splitting of aggreagated protein and the release of contents Please redraw the figure

sonication

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

After sonication zoom the tube instruct user to click on the tube for the contents display Animate the user interaction

After sonication the cells are lysed now serum contains high abundance proteins like albumin IgA IgG haptoglobin along with low abundance protein High abundance protein will interfere in gel separation and prevent the separation of low abundance proteins After sonication steps the sample is taken for further purification process

T3Sonication

Step 3

5

2

1

4

3

Purification steps same as IDD-6 Extraction of serum protein from Slide 17-32 For more information on the extraction steps go through future viewing IDD

Step 1 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Inoculation same as in IDD-1 Extraction of bacterial protein

Instruct user to open the lid of centrifuge and rotor Zoom in the rotor balance equal number of tubes inside the rotor Close the lid of rotor and of centrifuge with hand action Instruct user to set the rpm temperature and time parameters animate this in the display where user can increase and decrease the values of set parameters Animate the clock for 30min Kindly redraw the figures

Transfer the bacterial culture into the clean centrifuge tube under aseptic condition Centrifuge the culture for 10 min at 12000 rpm maintaining at 4rsquoC

Step 2 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

After 10min instruct user to open the lid of centrifuge rotor and animate the hand action to left the tube from rotor Now zoom the tube with pellet on bottom and liquid (supernatant) over it as shown in figure Now pipette out top portion (supernatant) completely into the discard the action should take place only when the user clicks on the pipette and the tube Kindly redraw the figures

Remove the supernatant completely without disturbing the pellet now take the pellet for further processing

Step 2 T2 Buffer treatment

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Show phosphate buffer bottle instruct user to pipette into the tube The user should click on the pipette for the action to be done Kindly redraw the figures

Wash the pellet with phosphate buffer thoroughly to remove the excess broth Once broth is removed completely cell lysis need to be carried out

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touches the solution in the tube Now display the screen of sonicator to make the necessary setup with help of user interactionuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5 sec 20 amplitude with 5 sec gap Sonication carries out cell lysis which helps for protein extraction

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves break open the cell wall and the contents are released into the buffer

High frequency sound waves

Zoom in a cell from the tube Show the sound waves hitting the cell and causing cell lyses and the release of contents Animate cell breaking with release of content into the tube from inside the cell Please redraw the figure

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Carry out the 3 centrifuge process as described earlier The user should click on ldquo Startrdquo for the centrifugation to on Please redraw the figure

Centrifuge the contents to remove the debris and collect the supernatant for further processing

Step 3

5

2

1

4

3

Extraction steps are same as IDD-1 Extraction of bacterial protein from Slide 16-32For more information on the purification steps go through future viewing IDD

Animation areaInteraction 1 slide-14 user carrying out sonication

without cooling on ice bath no gap between the bursts and proceeds further

Instructions let user place the tube on ice packs at the time of sonication and gives a time delay for few seconds between the bursts

Interaction 2 slide-14 user carrying out sonication for longer time

Instructions animate the tube getting hot and starts melting Excessive sonication will lead to heating and ice packs helps down in cooling the sample

Instructions Working area

Credits

Name of the sectionstage BACTERIAL CULTUREInteractivityarea

Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Button 01

Button 02

Button 03

Tab 01

Slide 4 Slide 7-10

Slide 5-6

Slide 11-12

Slide 14-17

Slide 13

QuestionnaireAPPENDIX

1

Question 1Amplitude used for the sonication of serum isa)20b)30c)40d)50

Answer 20

What is the purpose of sonication in bacterial protein extraction

a) To mix the cultureb) To denature the proteinc) To denature the

nucleotidesd) To lyse the cellsAnswer To lyse the cells

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

Step 3

5

2

1

4

3 Audio Narration (if any)

Description of the action interactivity

After sonication zoom the tube instruct user to click on the tube for the contents display Animate the user interaction

After sonication the cells are lysed now serum contains high abundance proteins like albumin IgA IgG haptoglobin along with low abundance protein High abundance protein will interfere in gel separation and prevent the separation of low abundance proteins After sonication steps the sample is taken for further purification process

T3Sonication

Step 3

5

2

1

4

3

Purification steps same as IDD-6 Extraction of serum protein from Slide 17-32 For more information on the extraction steps go through future viewing IDD

Step 1 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Inoculation same as in IDD-1 Extraction of bacterial protein

Instruct user to open the lid of centrifuge and rotor Zoom in the rotor balance equal number of tubes inside the rotor Close the lid of rotor and of centrifuge with hand action Instruct user to set the rpm temperature and time parameters animate this in the display where user can increase and decrease the values of set parameters Animate the clock for 30min Kindly redraw the figures

Transfer the bacterial culture into the clean centrifuge tube under aseptic condition Centrifuge the culture for 10 min at 12000 rpm maintaining at 4rsquoC

Step 2 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

After 10min instruct user to open the lid of centrifuge rotor and animate the hand action to left the tube from rotor Now zoom the tube with pellet on bottom and liquid (supernatant) over it as shown in figure Now pipette out top portion (supernatant) completely into the discard the action should take place only when the user clicks on the pipette and the tube Kindly redraw the figures

Remove the supernatant completely without disturbing the pellet now take the pellet for further processing

Step 2 T2 Buffer treatment

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Show phosphate buffer bottle instruct user to pipette into the tube The user should click on the pipette for the action to be done Kindly redraw the figures

Wash the pellet with phosphate buffer thoroughly to remove the excess broth Once broth is removed completely cell lysis need to be carried out

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touches the solution in the tube Now display the screen of sonicator to make the necessary setup with help of user interactionuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5 sec 20 amplitude with 5 sec gap Sonication carries out cell lysis which helps for protein extraction

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves break open the cell wall and the contents are released into the buffer

High frequency sound waves

Zoom in a cell from the tube Show the sound waves hitting the cell and causing cell lyses and the release of contents Animate cell breaking with release of content into the tube from inside the cell Please redraw the figure

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Carry out the 3 centrifuge process as described earlier The user should click on ldquo Startrdquo for the centrifugation to on Please redraw the figure

Centrifuge the contents to remove the debris and collect the supernatant for further processing

Step 3

5

2

1

4

3

Extraction steps are same as IDD-1 Extraction of bacterial protein from Slide 16-32For more information on the purification steps go through future viewing IDD

Animation areaInteraction 1 slide-14 user carrying out sonication

without cooling on ice bath no gap between the bursts and proceeds further

Instructions let user place the tube on ice packs at the time of sonication and gives a time delay for few seconds between the bursts

Interaction 2 slide-14 user carrying out sonication for longer time

Instructions animate the tube getting hot and starts melting Excessive sonication will lead to heating and ice packs helps down in cooling the sample

Instructions Working area

Credits

Name of the sectionstage BACTERIAL CULTUREInteractivityarea

Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Button 01

Button 02

Button 03

Tab 01

Slide 4 Slide 7-10

Slide 5-6

Slide 11-12

Slide 14-17

Slide 13

QuestionnaireAPPENDIX

1

Question 1Amplitude used for the sonication of serum isa)20b)30c)40d)50

Answer 20

What is the purpose of sonication in bacterial protein extraction

a) To mix the cultureb) To denature the proteinc) To denature the

nucleotidesd) To lyse the cellsAnswer To lyse the cells

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

Step 3

5

2

1

4

3

Purification steps same as IDD-6 Extraction of serum protein from Slide 17-32 For more information on the extraction steps go through future viewing IDD

Step 1 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Inoculation same as in IDD-1 Extraction of bacterial protein

Instruct user to open the lid of centrifuge and rotor Zoom in the rotor balance equal number of tubes inside the rotor Close the lid of rotor and of centrifuge with hand action Instruct user to set the rpm temperature and time parameters animate this in the display where user can increase and decrease the values of set parameters Animate the clock for 30min Kindly redraw the figures

Transfer the bacterial culture into the clean centrifuge tube under aseptic condition Centrifuge the culture for 10 min at 12000 rpm maintaining at 4rsquoC

Step 2 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

After 10min instruct user to open the lid of centrifuge rotor and animate the hand action to left the tube from rotor Now zoom the tube with pellet on bottom and liquid (supernatant) over it as shown in figure Now pipette out top portion (supernatant) completely into the discard the action should take place only when the user clicks on the pipette and the tube Kindly redraw the figures

Remove the supernatant completely without disturbing the pellet now take the pellet for further processing

Step 2 T2 Buffer treatment

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Show phosphate buffer bottle instruct user to pipette into the tube The user should click on the pipette for the action to be done Kindly redraw the figures

Wash the pellet with phosphate buffer thoroughly to remove the excess broth Once broth is removed completely cell lysis need to be carried out

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touches the solution in the tube Now display the screen of sonicator to make the necessary setup with help of user interactionuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5 sec 20 amplitude with 5 sec gap Sonication carries out cell lysis which helps for protein extraction

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves break open the cell wall and the contents are released into the buffer

High frequency sound waves

Zoom in a cell from the tube Show the sound waves hitting the cell and causing cell lyses and the release of contents Animate cell breaking with release of content into the tube from inside the cell Please redraw the figure

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Carry out the 3 centrifuge process as described earlier The user should click on ldquo Startrdquo for the centrifugation to on Please redraw the figure

Centrifuge the contents to remove the debris and collect the supernatant for further processing

Step 3

5

2

1

4

3

Extraction steps are same as IDD-1 Extraction of bacterial protein from Slide 16-32For more information on the purification steps go through future viewing IDD

Animation areaInteraction 1 slide-14 user carrying out sonication

without cooling on ice bath no gap between the bursts and proceeds further

Instructions let user place the tube on ice packs at the time of sonication and gives a time delay for few seconds between the bursts

Interaction 2 slide-14 user carrying out sonication for longer time

Instructions animate the tube getting hot and starts melting Excessive sonication will lead to heating and ice packs helps down in cooling the sample

Instructions Working area

Credits

Name of the sectionstage BACTERIAL CULTUREInteractivityarea

Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Button 01

Button 02

Button 03

Tab 01

Slide 4 Slide 7-10

Slide 5-6

Slide 11-12

Slide 14-17

Slide 13

QuestionnaireAPPENDIX

1

Question 1Amplitude used for the sonication of serum isa)20b)30c)40d)50

Answer 20

What is the purpose of sonication in bacterial protein extraction

a) To mix the cultureb) To denature the proteinc) To denature the

nucleotidesd) To lyse the cellsAnswer To lyse the cells

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

Step 1 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Inoculation same as in IDD-1 Extraction of bacterial protein

Instruct user to open the lid of centrifuge and rotor Zoom in the rotor balance equal number of tubes inside the rotor Close the lid of rotor and of centrifuge with hand action Instruct user to set the rpm temperature and time parameters animate this in the display where user can increase and decrease the values of set parameters Animate the clock for 30min Kindly redraw the figures

Transfer the bacterial culture into the clean centrifuge tube under aseptic condition Centrifuge the culture for 10 min at 12000 rpm maintaining at 4rsquoC

Step 2 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

After 10min instruct user to open the lid of centrifuge rotor and animate the hand action to left the tube from rotor Now zoom the tube with pellet on bottom and liquid (supernatant) over it as shown in figure Now pipette out top portion (supernatant) completely into the discard the action should take place only when the user clicks on the pipette and the tube Kindly redraw the figures

Remove the supernatant completely without disturbing the pellet now take the pellet for further processing

Step 2 T2 Buffer treatment

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Show phosphate buffer bottle instruct user to pipette into the tube The user should click on the pipette for the action to be done Kindly redraw the figures

Wash the pellet with phosphate buffer thoroughly to remove the excess broth Once broth is removed completely cell lysis need to be carried out

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touches the solution in the tube Now display the screen of sonicator to make the necessary setup with help of user interactionuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5 sec 20 amplitude with 5 sec gap Sonication carries out cell lysis which helps for protein extraction

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves break open the cell wall and the contents are released into the buffer

High frequency sound waves

Zoom in a cell from the tube Show the sound waves hitting the cell and causing cell lyses and the release of contents Animate cell breaking with release of content into the tube from inside the cell Please redraw the figure

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Carry out the 3 centrifuge process as described earlier The user should click on ldquo Startrdquo for the centrifugation to on Please redraw the figure

Centrifuge the contents to remove the debris and collect the supernatant for further processing

Step 3

5

2

1

4

3

Extraction steps are same as IDD-1 Extraction of bacterial protein from Slide 16-32For more information on the purification steps go through future viewing IDD

Animation areaInteraction 1 slide-14 user carrying out sonication

without cooling on ice bath no gap between the bursts and proceeds further

Instructions let user place the tube on ice packs at the time of sonication and gives a time delay for few seconds between the bursts

Interaction 2 slide-14 user carrying out sonication for longer time

Instructions animate the tube getting hot and starts melting Excessive sonication will lead to heating and ice packs helps down in cooling the sample

Instructions Working area

Credits

Name of the sectionstage BACTERIAL CULTUREInteractivityarea

Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Button 01

Button 02

Button 03

Tab 01

Slide 4 Slide 7-10

Slide 5-6

Slide 11-12

Slide 14-17

Slide 13

QuestionnaireAPPENDIX

1

Question 1Amplitude used for the sonication of serum isa)20b)30c)40d)50

Answer 20

What is the purpose of sonication in bacterial protein extraction

a) To mix the cultureb) To denature the proteinc) To denature the

nucleotidesd) To lyse the cellsAnswer To lyse the cells

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

Step 2 T1 Sample preparation (bacterial)

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

After 10min instruct user to open the lid of centrifuge rotor and animate the hand action to left the tube from rotor Now zoom the tube with pellet on bottom and liquid (supernatant) over it as shown in figure Now pipette out top portion (supernatant) completely into the discard the action should take place only when the user clicks on the pipette and the tube Kindly redraw the figures

Remove the supernatant completely without disturbing the pellet now take the pellet for further processing

Step 2 T2 Buffer treatment

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Show phosphate buffer bottle instruct user to pipette into the tube The user should click on the pipette for the action to be done Kindly redraw the figures

Wash the pellet with phosphate buffer thoroughly to remove the excess broth Once broth is removed completely cell lysis need to be carried out

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touches the solution in the tube Now display the screen of sonicator to make the necessary setup with help of user interactionuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5 sec 20 amplitude with 5 sec gap Sonication carries out cell lysis which helps for protein extraction

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves break open the cell wall and the contents are released into the buffer

High frequency sound waves

Zoom in a cell from the tube Show the sound waves hitting the cell and causing cell lyses and the release of contents Animate cell breaking with release of content into the tube from inside the cell Please redraw the figure

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Carry out the 3 centrifuge process as described earlier The user should click on ldquo Startrdquo for the centrifugation to on Please redraw the figure

Centrifuge the contents to remove the debris and collect the supernatant for further processing

Step 3

5

2

1

4

3

Extraction steps are same as IDD-1 Extraction of bacterial protein from Slide 16-32For more information on the purification steps go through future viewing IDD

Animation areaInteraction 1 slide-14 user carrying out sonication

without cooling on ice bath no gap between the bursts and proceeds further

Instructions let user place the tube on ice packs at the time of sonication and gives a time delay for few seconds between the bursts

Interaction 2 slide-14 user carrying out sonication for longer time

Instructions animate the tube getting hot and starts melting Excessive sonication will lead to heating and ice packs helps down in cooling the sample

Instructions Working area

Credits

Name of the sectionstage BACTERIAL CULTUREInteractivityarea

Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Button 01

Button 02

Button 03

Tab 01

Slide 4 Slide 7-10

Slide 5-6

Slide 11-12

Slide 14-17

Slide 13

QuestionnaireAPPENDIX

1

Question 1Amplitude used for the sonication of serum isa)20b)30c)40d)50

Answer 20

What is the purpose of sonication in bacterial protein extraction

a) To mix the cultureb) To denature the proteinc) To denature the

nucleotidesd) To lyse the cellsAnswer To lyse the cells

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

Step 2 T2 Buffer treatment

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Show phosphate buffer bottle instruct user to pipette into the tube The user should click on the pipette for the action to be done Kindly redraw the figures

Wash the pellet with phosphate buffer thoroughly to remove the excess broth Once broth is removed completely cell lysis need to be carried out

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touches the solution in the tube Now display the screen of sonicator to make the necessary setup with help of user interactionuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5 sec 20 amplitude with 5 sec gap Sonication carries out cell lysis which helps for protein extraction

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves break open the cell wall and the contents are released into the buffer

High frequency sound waves

Zoom in a cell from the tube Show the sound waves hitting the cell and causing cell lyses and the release of contents Animate cell breaking with release of content into the tube from inside the cell Please redraw the figure

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Carry out the 3 centrifuge process as described earlier The user should click on ldquo Startrdquo for the centrifugation to on Please redraw the figure

Centrifuge the contents to remove the debris and collect the supernatant for further processing

Step 3

5

2

1

4

3

Extraction steps are same as IDD-1 Extraction of bacterial protein from Slide 16-32For more information on the purification steps go through future viewing IDD

Animation areaInteraction 1 slide-14 user carrying out sonication

without cooling on ice bath no gap between the bursts and proceeds further

Instructions let user place the tube on ice packs at the time of sonication and gives a time delay for few seconds between the bursts

Interaction 2 slide-14 user carrying out sonication for longer time

Instructions animate the tube getting hot and starts melting Excessive sonication will lead to heating and ice packs helps down in cooling the sample

Instructions Working area

Credits

Name of the sectionstage BACTERIAL CULTUREInteractivityarea

Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Button 01

Button 02

Button 03

Tab 01

Slide 4 Slide 7-10

Slide 5-6

Slide 11-12

Slide 14-17

Slide 13

QuestionnaireAPPENDIX

1

Question 1Amplitude used for the sonication of serum isa)20b)30c)40d)50

Answer 20

What is the purpose of sonication in bacterial protein extraction

a) To mix the cultureb) To denature the proteinc) To denature the

nucleotidesd) To lyse the cellsAnswer To lyse the cells

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Instruct user to place the tube on ice with cap open Show the sonicator instrument place the tube such that the tip of sonicator rod touches the solution in the tube Now display the screen of sonicator to make the necessary setup with help of user interactionuser should click on the sonicator to proceed with sonication

Keep the sample on ice and start sonication by providing 6 cycles of pulses for 5 sec 20 amplitude with 5 sec gap Sonication carries out cell lysis which helps for protein extraction

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves break open the cell wall and the contents are released into the buffer

High frequency sound waves

Zoom in a cell from the tube Show the sound waves hitting the cell and causing cell lyses and the release of contents Animate cell breaking with release of content into the tube from inside the cell Please redraw the figure

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Carry out the 3 centrifuge process as described earlier The user should click on ldquo Startrdquo for the centrifugation to on Please redraw the figure

Centrifuge the contents to remove the debris and collect the supernatant for further processing

Step 3

5

2

1

4

3

Extraction steps are same as IDD-1 Extraction of bacterial protein from Slide 16-32For more information on the purification steps go through future viewing IDD

Animation areaInteraction 1 slide-14 user carrying out sonication

without cooling on ice bath no gap between the bursts and proceeds further

Instructions let user place the tube on ice packs at the time of sonication and gives a time delay for few seconds between the bursts

Interaction 2 slide-14 user carrying out sonication for longer time

Instructions animate the tube getting hot and starts melting Excessive sonication will lead to heating and ice packs helps down in cooling the sample

Instructions Working area

Credits

Name of the sectionstage BACTERIAL CULTUREInteractivityarea

Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Button 01

Button 02

Button 03

Tab 01

Slide 4 Slide 7-10

Slide 5-6

Slide 11-12

Slide 14-17

Slide 13

QuestionnaireAPPENDIX

1

Question 1Amplitude used for the sonication of serum isa)20b)30c)40d)50

Answer 20

What is the purpose of sonication in bacterial protein extraction

a) To mix the cultureb) To denature the proteinc) To denature the

nucleotidesd) To lyse the cellsAnswer To lyse the cells

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

High frequency sound waves break open the cell wall and the contents are released into the buffer

High frequency sound waves

Zoom in a cell from the tube Show the sound waves hitting the cell and causing cell lyses and the release of contents Animate cell breaking with release of content into the tube from inside the cell Please redraw the figure

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Carry out the 3 centrifuge process as described earlier The user should click on ldquo Startrdquo for the centrifugation to on Please redraw the figure

Centrifuge the contents to remove the debris and collect the supernatant for further processing

Step 3

5

2

1

4

3

Extraction steps are same as IDD-1 Extraction of bacterial protein from Slide 16-32For more information on the purification steps go through future viewing IDD

Animation areaInteraction 1 slide-14 user carrying out sonication

without cooling on ice bath no gap between the bursts and proceeds further

Instructions let user place the tube on ice packs at the time of sonication and gives a time delay for few seconds between the bursts

Interaction 2 slide-14 user carrying out sonication for longer time

Instructions animate the tube getting hot and starts melting Excessive sonication will lead to heating and ice packs helps down in cooling the sample

Instructions Working area

Credits

Name of the sectionstage BACTERIAL CULTUREInteractivityarea

Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Button 01

Button 02

Button 03

Tab 01

Slide 4 Slide 7-10

Slide 5-6

Slide 11-12

Slide 14-17

Slide 13

QuestionnaireAPPENDIX

1

Question 1Amplitude used for the sonication of serum isa)20b)30c)40d)50

Answer 20

What is the purpose of sonication in bacterial protein extraction

a) To mix the cultureb) To denature the proteinc) To denature the

nucleotidesd) To lyse the cellsAnswer To lyse the cells

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

Step 3 T3 Sonication

5

2

1

4

3 Audio Narration (if any)Description of the action interactivity

Carry out the 3 centrifuge process as described earlier The user should click on ldquo Startrdquo for the centrifugation to on Please redraw the figure

Centrifuge the contents to remove the debris and collect the supernatant for further processing

Step 3

5

2

1

4

3

Extraction steps are same as IDD-1 Extraction of bacterial protein from Slide 16-32For more information on the purification steps go through future viewing IDD

Animation areaInteraction 1 slide-14 user carrying out sonication

without cooling on ice bath no gap between the bursts and proceeds further

Instructions let user place the tube on ice packs at the time of sonication and gives a time delay for few seconds between the bursts

Interaction 2 slide-14 user carrying out sonication for longer time

Instructions animate the tube getting hot and starts melting Excessive sonication will lead to heating and ice packs helps down in cooling the sample

Instructions Working area

Credits

Name of the sectionstage BACTERIAL CULTUREInteractivityarea

Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Button 01

Button 02

Button 03

Tab 01

Slide 4 Slide 7-10

Slide 5-6

Slide 11-12

Slide 14-17

Slide 13

QuestionnaireAPPENDIX

1

Question 1Amplitude used for the sonication of serum isa)20b)30c)40d)50

Answer 20

What is the purpose of sonication in bacterial protein extraction

a) To mix the cultureb) To denature the proteinc) To denature the

nucleotidesd) To lyse the cellsAnswer To lyse the cells

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

Step 3

5

2

1

4

3

Extraction steps are same as IDD-1 Extraction of bacterial protein from Slide 16-32For more information on the purification steps go through future viewing IDD

Animation areaInteraction 1 slide-14 user carrying out sonication

without cooling on ice bath no gap between the bursts and proceeds further

Instructions let user place the tube on ice packs at the time of sonication and gives a time delay for few seconds between the bursts

Interaction 2 slide-14 user carrying out sonication for longer time

Instructions animate the tube getting hot and starts melting Excessive sonication will lead to heating and ice packs helps down in cooling the sample

Instructions Working area

Credits

Name of the sectionstage BACTERIAL CULTUREInteractivityarea

Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Button 01

Button 02

Button 03

Tab 01

Slide 4 Slide 7-10

Slide 5-6

Slide 11-12

Slide 14-17

Slide 13

QuestionnaireAPPENDIX

1

Question 1Amplitude used for the sonication of serum isa)20b)30c)40d)50

Answer 20

What is the purpose of sonication in bacterial protein extraction

a) To mix the cultureb) To denature the proteinc) To denature the

nucleotidesd) To lyse the cellsAnswer To lyse the cells

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

Animation areaInteraction 1 slide-14 user carrying out sonication

without cooling on ice bath no gap between the bursts and proceeds further

Instructions let user place the tube on ice packs at the time of sonication and gives a time delay for few seconds between the bursts

Interaction 2 slide-14 user carrying out sonication for longer time

Instructions animate the tube getting hot and starts melting Excessive sonication will lead to heating and ice packs helps down in cooling the sample

Instructions Working area

Credits

Name of the sectionstage BACTERIAL CULTUREInteractivityarea

Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Button 01

Button 02

Button 03

Tab 01

Slide 4 Slide 7-10

Slide 5-6

Slide 11-12

Slide 14-17

Slide 13

QuestionnaireAPPENDIX

1

Question 1Amplitude used for the sonication of serum isa)20b)30c)40d)50

Answer 20

What is the purpose of sonication in bacterial protein extraction

a) To mix the cultureb) To denature the proteinc) To denature the

nucleotidesd) To lyse the cellsAnswer To lyse the cells

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

QuestionnaireAPPENDIX

1

Question 1Amplitude used for the sonication of serum isa)20b)30c)40d)50

Answer 20

What is the purpose of sonication in bacterial protein extraction

a) To mix the cultureb) To denature the proteinc) To denature the

nucleotidesd) To lyse the cellsAnswer To lyse the cells

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

QuestionnaireAPPENDIX

1

What is the purpose of sonication in serum protein extraction

a) To mix the cultureb) To deplete high abundance proteinc) To denature the nucleotidesd) To break the protein aggregatesAnswer To break the protein aggregates

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

Links for further readingAPPENDIX

2

Chen JH Chang YW Yao CW et al Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometryProc Natl Acad Sci U S A2004 7101(49)17039-44

Eymann C Dreisbach A Albrecht D A comprehensive proteome map of growing Bacillus subtilis cells

Proteomics 2004 2849-76

Maldonado AM Echevarriacutea-Zomentildeo S Jean-Baptiste S et al Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis Proteomics 2008 71(4)461-72

2DE Tutorials by Angelika Goumlrg httpwwwwzwtumdeblmdeg BOOKS Biochemistry by Stryer et al 5th editionBiochemistry by ALLehninger et al 3rd editionBiochemistry by Voet amp Voet 3rd edition

SummaryAPPENDIX

3

Ultrasonic waves generated by a sonicator lyse cells through shear forcesComplete shearing is obtained when maximal agitation is achieved but caremust be taken to minimize heating and foaming

Sonicate cell suspension in short bursts to avoid heatingCool on ice between bursts Sonication is a vigorous way of cell lysis

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• Step 2
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• Step 1
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