sds-page part ii
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Electrophoresis and Visualization. SDS-Page Part II. Boiling Lysates. Proteins are being denatured by the combination of SDS and heat. Resultant proteins take on a rod-like shape and a uniform negative charge-to-mass ratio proportional to their molecular weight. Electrophoresis. - PowerPoint PPT PresentationTRANSCRIPT
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Electrophoresis and VisualizationElectrophoresis and Visualization
SDS-Page Part IISDS-Page Part II
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Boiling LysatesBoiling Lysates Resultant proteins take on a rod-like
shape and a uniform negative charge-to-mass ratio proportional to their molecular weight
Resultant proteins take on a rod-like shape and a uniform negative charge-to-mass ratio proportional to their molecular weight
Proteins are being denatured by the combination of SDS and heat
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ElectrophoresisElectrophoresis Use polyacrylamide gel
Stacking gel- Large pore size and stacks proteins Separating gel- Small pore size and Separates
proteins base on molecular weight Why acrylamide?
Acrylamide gel: tight matrix that is ideal for protein separation
Smaller pore size than agarose Proteins are much smaller than intact
chromosomal DNA
Use polyacrylamide gel Stacking gel- Large pore size and stacks proteins Separating gel- Small pore size and Separates
proteins base on molecular weight Why acrylamide?
Acrylamide gel: tight matrix that is ideal for protein separation
Smaller pore size than agarose Proteins are much smaller than intact
chromosomal DNA
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How does an SDS-PAGE gel work?
How does an SDS-PAGE gel work?
Negatively charged proteins move to the positive electrode
Smaller proteins move faster
Proteins separate by size
Negatively charged proteins move to the positive electrode
Smaller proteins move faster
Proteins separate by size
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-
+
s-s
SDS, heat
proteins with SDS
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The SDS-PAGE apparatusThe SDS-PAGE apparatus
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Tracking the samplesTracking the samples You will monitor the
progress of electrophoresis to ensure your samples don’t run off the gel Using
Bromophenol Blue that is in the sample buffer
Refer to next slide
You will monitor the progress of electrophoresis to ensure your samples don’t run off the gel Using
Bromophenol Blue that is in the sample buffer
Refer to next slide
What is in the sample Buffer?*Tris buffer to provide appropriate pH*SDS (sodium dodecyl sulphate) detergent to dissolve proteins and give them a negative charge*Glycerol to make samples sink into wells*Bromophenol Blue dye to visualize samples
What is in the sample Buffer?*Tris buffer to provide appropriate pH*SDS (sodium dodecyl sulphate) detergent to dissolve proteins and give them a negative charge*Glycerol to make samples sink into wells*Bromophenol Blue dye to visualize samples
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VisualizingVisualizing When electrophoresis is complete,
you will stain with Coomassie Blue and Destain The stain and destain contains
methanol and acetic acid that helps to fix the proteins in the gel.
Coomassie Blue binds to proteins
When electrophoresis is complete, you will stain with Coomassie Blue and Destain The stain and destain contains
methanol and acetic acid that helps to fix the proteins in the gel.
Coomassie Blue binds to proteins
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What next?What next?Obtain information about the markerObtain information about the marker
Obtain a picture of your gelObtain a picture of your gel
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Calculate Rf Value and GraphCalculate Rf Value and Graph
Dye front
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Calculating Rf valuesCalculating Rf values Calculating Rf values
Rf = distance migrated by the moleculedistance migrated by the dye front
Plot a standard curve on a log graph paper or regular graph paper. You must convert to log10(MW of proteins in the
marker if you use regular graph paper) Use your curve in the identifying the unknown and
answer the questions in your lab manual
Calculating Rf values
Rf = distance migrated by the moleculedistance migrated by the dye front
Plot a standard curve on a log graph paper or regular graph paper. You must convert to log10(MW of proteins in the
marker if you use regular graph paper) Use your curve in the identifying the unknown and
answer the questions in your lab manual
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kDa Rf203 8.5 135 12.0
86 18.5
19 41.5
33 34.0
8 44.5
41 28.0
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CautionCaution Sample buffer waste- contains a
hazardous reducing agent, Beta mercaptoethanol -stinks like rotten eggs Eppendorf tubes should be thrown
into the sample buffer waste container
Sample buffer waste- contains a hazardous reducing agent, Beta mercaptoethanol -stinks like rotten eggs Eppendorf tubes should be thrown
into the sample buffer waste container
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Lab summaryLab summary Don’t forget to include the protocol
from Part I! Answer all the questions Make sure to include tables (of both
the standard measurements and unknown measurements) and your graph!
Conclusions?
Don’t forget to include the protocol from Part I!
Answer all the questions Make sure to include tables (of both
the standard measurements and unknown measurements) and your graph!
Conclusions?