Experimental Design                                                         

Fig1: Two methods for carrying out differential protein expression analysis: A) 2-D gel electrophoresis method – After sample proteins are extracted and quantified, electrophoresis is carried out in both the dimensions and resulting gels are stained and scanned. Scanned images are then analyzed using IMP7 software. B) 2D-DIGE method – Samples are labeled with CyDye and mixed together before rehydrating the IPG strips. Once the proteins are separated on the basis of both their isoelectric points and molecular weights, the gels are scanned and analysed using DeCyder software to study the differential protein expression patterns.

SECTION I : Differential protein expression analysis using 2-D gel electrophoresis

Protocol: 

1. Protein isolation:

            Materials provided: Normal and tumor tissue samples

Materials required: Scalpel, mortar and pestle, autoclaved tubes and tips

Reagents: Liquid nitrogen, Ethanol, Trizol, chloroform, acetone, guanidium hydrochloride, urea, thiourea, CHAPS, IPG buffer (pH 4-7), bromophenol blue

Instruments Required: Laminar Air-flow, centrifuge, -80oC freezer, weighing balance, sonicator

a. After surgical resection, snap freeze the sample using liquid nitrogen and immediately

store at   -80oC 

b. Weigh 50 mg of tissue and wash it twice with 1X PBS (pH 7.3)

c. Sonicate the sample for a total of 15 cycles using the following settings: Amplitude: 40%;

Pulse on for 10 sec., gap of 10 sec.

d. Centrifuge the sample at 14,000 rpm for 15 min. at 40C temperature

e. Collect the supernatant in a fresh tube

f. Add 1 ml trizol reagent to this supernatent.

g. Immediately add 200 ul chloroform to the same mixture, shake vigorously for 15 sec &

incubate for 15 min at RT.

h. Centrifuge at 12,000 g for 15 min. Bacterial sample gets separated into three distinct

layers.

i. Carefully remove upper transparent layer containing RNA using a micropipette.

j. To the bottom two layers, add 300 ul ethanol, incubate at RT for 3 min and centrifuge at

2,000 x g for 5 min to remove DNA.

k. Separate the supernatant containing protein collect into a fresh tube. Retain and store the

DNA pellet at – 20°C.

l. To the supernatant, add 4 volumes of chilled acetone (acetone kept in – 20°C at least for

4 h) and incubate for 6 h at – 20°C.

m. After incubation centrifuge at 12,000 x g for 5 min.

n. Discard the supernatant, retain the pellet of protein.

o. Wash the protein pellet with 95% (95% ethanol+5% water) ethanol or acetone (4 times).

p. After each wash give a brief spin to settle proteins, discarding the supernatant each time.

q. Dry the pellet in the room temperature.

r. Reconstitute the dried pellet in buffer containing 8M Urea, 2M Thiourea, 2% CHAPS,

2% IPG buffer and 0.002% Bromophenol Blue and store the protein solution at – 20°C

till further use.

NOTE:- An outline of further steps involved in 2-D gel electrophoresis is given below.

Details of these steps are presented in Experiment 1. The reader is requested to go through

the Procedure section of Experiment 1 for a recap of the techniques involved.

2. Protein Quantitation:

Proteins are quantified by the Bradford method using the principle of shift in absorbance maxima of the dye in the presence of proteins.

3. First Dimensional - Iso-Electric Focusing (IEF):

This involves the following steps:-

A. Rehydration of IPG strips – Samples are loaded onto the IPG stripsB. Focusing of IPG strips – Proteins are separated on the basis of their pI

C. Equilibration of strips – Separated proteins are further denatured and saturated with SDS

4. Second dimension - SDS-PAGE:

This involves the following steps:-

A. Preparation of solutions – An important step to ensure a proper runB. Gel casting – The solutions prepared are then mixed and poured into the casting unit.

The gel is allowed to polymerize between the glass plates placed in the casting unit.C. Gel run – Proteins are separated on the basis of their molecular weight

5. Staining and Scanning of 2-D electrophoresis gels:

The the following steps are involved:-

A. Preparation of solutions B. Staining and destaining of the gels – The gels are stained with Coomassie Brilliant

Blue dye for 5-6 hours on a shaker. Gels are then destained in a similar manner to be able to visualize the proteins as blue coloured spots against a transparent background

C. Scanning the gels – This is done with the help of the ImageScanner III LabScan 6.0. Images so obtained can be used for further analysis

6. Image analysis:

The ImageMaster 2D Platinum 7.0 software is used to find the total number of spots on the

gels and to identify the spots that are differentially expressed between the normal and tumor

tissue sample. By applying various statistical analysis tools to the data obtained from this

software it is possible to locate the spots that are up- or down-regulated in the diseased state

as compared to the normal tissue The fold change by which the proteins are differentially

expressed can also derived. Details of the steps followed during the analysis are given

below:-

6A) Image processing

1. Create a project and load the control and treated gel images in folder for analysis. Once the gels are loaded cropping needs to be carried out.

Fig. 2: Importing gel images into master file.

2. Rotate the images to align all the gels in same orientation. Such image processing does not affect original image files. The bold horizontal grid line acts as a landmark to help visualize the rotation. Flip icon is used when gel images are scanned in the wrong direction. Gel images can be flipped Horizontally or Vertically to produce their correct mirror image.

3. Crop the gel images with the Crop tool. This creates new gels that only contain the selected area and removes the outer area. Once the gels are cropped the images are ready for the analysis.

FLip icon

CROP icon

6B) Spot processing

1. Once gels have been added to a project and user can have a good look at them, gels are ready for spot detection. A spot delineates a small region in the gel where protein is present. This shape is automatically differentiated by a spot detection algorithm and quantified and its intensity, area and volume are computed.

2. To detect the spots the optimum spot detection parameters need to be defined. The parameter smooth helps to detect all real spots and split the overlapping ones. Subsequently, Saliency and Min Area values helps out to filter the noise.

3. After spot detection, zoom each and every spots to check for a real spot. The software detects dusts, artifacts, which needs to removed from analysis.

4. User must detect artifacts from the detected spots which needs to be removed from analysis. If user finds unwanted spot, they can be deleated and if spots are found at exactly the position across the gels, such spots can be landmarked. Land mark of spots helps in matching. Once land marking is over, save the gels and import the gels into Matching folder.

6C) Gel matching

1. Matching of gels must be carried out after landmarking. Matching algorithm first matches the landmarked spots, then matches the nearby spots. In case if less number of matches are produced, create few more landmarks and try again. Landmarking must be in such a way that it should cover entire gel area.

2. Vector lines helps to check the matching process, as to how correctly the gels have matched with each other. If the vector lines are of same length and in same direction, the matching is said to be perfect. If the vector lines are all of different sizes and in different direction, we say matching is not perfect.

3. The overlay option helps user to check the profile of each gel, to detect matching pattern.

.

Once overlay is done, and user is satisfied with matching user can proceed with the data analysis if not he can again restart the matching. Now save the workspace and drag the images in to classes folder.

6D) Statistical data analysis

1. Click open the gels from classes folder. Select Edit:All spots:Spot ID display. Select Report:Spot table. A table with values appears at the bottom of the window.

2. Spots across the gel can be selected and compared to know their pI, volume, intensity, MW etc.

3. The protein spots can be represented in the 3-D form with peak height denotes its intensity and can be rotated to view in different angles. This helps user to make a rough calculation for the fold difference expression of protein between the samples. To make accurate calculation for fold difference, user must do the statistical analysis for the data..

4. Click on the statistical analysis table to get statistical information of the spots. The data can be used to produce fold difference between the spots, to determine increased and decreased spots, to generate a histogram for distribution of spots and calculate t-test , ANOVA , other statistical analysis.

SECTION II : Differential protein expression analysis using 2D-DIGE

NOTE:- An outline of steps that are common to 2-D gel electrophoresis and 2D-DIGE are given below. Details of all other steps are presented in this section.

1. Protein isolation:

Tissue samples are washed with PBS buffer and sonicated to get the whole cell lysate. The

supernatant is then treated with Trizol reagent to get a pure preparation of proteins, devoid of any

impurities.

2. Protein Quantitation:

Proteins are quantified by the Bradford method using the principle of shift in absorbance maxima

of the dye in the presence of proteins.

3. Sample preparation:

Materials provided: CyDye minimal labeling kit

Materials required: Sample, pipette, eppendorf tubes, ice and tips.

Reagents: DMF.

Instrument required: Mini centrifuge and bench top vortex.

3A.     Preparation of CyDye

CyDye DIGE Fluor minimal dyes need to be reconstituted in anhydrous DMF of high-quality

(99.8% pure) to give a concentration of 1 nmol/uL. To avoid contamination, DMF and should be

replaced at least every 3 months.

a.  Stock Solution: Add 25ul of DMF to 25nmol of CyDye Fluor, vortex and centrifuge it to

collect the reconstituted dye. This concentrated stock solution can be kept at -20°C for upto

three months.

b. Working Solution:  Add 2ul of stock solution to 3ul of DMF, vortex, spin, store it at -20°C

for further use.

3B.     Sample labeling

a. Thaw the protein samples and adjust their pH to 8.5.

b. Add 1ul of working dye solution to 50ug of protein. Follow the same step for each

sample with its respective dye, as per the experimental design given in Table 1.

c. Mix by vortexing. Incubate on ice for 30min in the dark room.

d. Add 1ul of 10mM lysine to stop the labeling reaction. Mix well by vortexing and keep

on ice for 10min in the dark.

e. After 10min, the labeled sample can be kept at -70°C in dark upto 3 months for further

use or can be mixed into single tube and combined with Rehydration buffer to carry out

IEF.

Experimental design: 1

Control Test Internal Standard GEL

(50ug) (50ug) (5+5)ug Concentration

Cy5 Cy3 Cy2 Dye

C1 T1 C1-C5 +T1-T5 GEL1

C2 T2 C1-C5 +T1-T5 GEL2

C3 T3 C1-C5 +T1-T5 GEL3

C4 T4 C1-C5 +T1-T5 GEL4

C5 T5 C1-C5 +T1-T5 GEL5

Experimental design: 2

Sample Stage Internal Standard GEL

(50ug) (50ug) (25+25)ug Concentration

Cy5 Cy3 Cy2 Dye

S1 S2 S1+S2 GEL1

S2 S3 S1+S3 GEL2

S3 S4 S1+S4 GEL3

Experimental design: 3

Sample Stage Internal Standard GEL

(50ug) (50ug) (25+25)ug Concentration

Cy5 Cy3 Cy2 Dye

S1 S2 S1+S2 GEL1

S1 S3 S1+S3 GEL2

S1 S4 S1+S4 GEL3

S2 S3 S2+S3 GEL4

S2 S4 S2+S4 GEL5

S3 S4 S3+S4 GEL6

Table 1: Cy dye labeling: Three sample experimental designs for rehydrating a 24cms IPGstrip

are presented. Protein loading capacity for each sample is 50ug. Samples are distributed to get

labeled with Cy5 and Cy3 and the pool of both control and test samples are labeled with Cy2.

4. First Dimensional - Iso-Electric Focusing (IEF):

This involves the following steps:-

A. Rehydration of IPG strips – Samples are loaded onto the IPG strips

B. Focusing of IPG strips – Proteins are separated on the basis of their pI

C. Equilibration of strips – Separated proteins are further denatured and saturated with SDS

5. Second dimension - SDS-PAGE:

This involves the following steps:-

A. Preparation of solutions – An important step to ensure a proper run

B. Gel casting – The solutions prepared are then mixed and poured into the casting unit.

The gel is allowed to polymerize between the glass plates placed in the casting unit.

C. Gel run – Proteins are separated on the basis of their molecular weight

Carefully remove the plates from the electrophoresis assembly and keep the gels between the

glass plates. If required, store the gels in SDS electrophoresis running buffer at +4oC in the dark

for not more than a day. It is best to use the gels immediately for scanning.

6. Scanning of 2D-DIGE gels:

Materials provided: Typhoon Imager.

Materials required: lint free tissue, distilled water.

6A. Starting the Imager

a.    Turn on the scanner and workstation. The POWER light turns on and remains red

during the self-test sequence (Fig.2). A green light flashes during scanner

initialization step and later, a steady green light glows which indicates that the

scanner is ready for scanning.  Let the instrument warm up for 30 min before

scanning any gel.

Fig. 2: Schematic representation of Typhoon imager.

b.   After the power indicator light turns solid green, click on Typhoon Imager icon on

the workstation desktop to start the Scanner software.

c.    The Typhoon scanner control window appears (Fig. 3)

Fig. 3: Image of the Typhoon scanner control window.

6B. Prepare the Cassette for a scan

a.    After the 2D-DIGE run, take out each gel plate from the 2D running system,

clean it properly with distilled water using a lint free paper. Keep the glass plate

over the plate stand for drying.

b.   Clean the glass platen and sample lid with 10% hydrogen peroxide and distilled

water before placing the gel on the platen.

c.    After the glass plate is dried and cleaned, place the shorter glass side facing down

on the platen. Close the sample lid of the scanner.   

6C. Set up a scan

In the Scanner software, already opened on the workstation desktop, choose the following

options in the Setup Area window:-

i. Use TRAY EDITOR window to create a separate scan area, with a name,

for each sample.

ii. Select the parameters to be saved with the template in the SETUP AREA

of the Scanner Control window (Fig. 3)

iii. Click SETUP button and select the parameters for fluorescence scan

iv. Choose SAVE AS TEMPLATE from the “Templates” menu and type a

new name for the template in the box that appears.

Or, choose LOAD from the “Templates” menu and select template from

the list.

6D. Scan a sample

a. To scan a single gel:-

i. Ensure correct parameters are selected in Scanner Control and

Fluorescence Setup windows

ii. Click SCAN

iii. Type name in the Save AS: File name

iv. Click SAVE to start the scan

b. To scan multiple gels:-

i. Ensure correct parameters are selected in Scanner Control and

Fluorescence Setup windows

ii. Select DIGE FILE NAMING FORMAT check box.

iii. Click SCAN button

iv. Select FOLDER in Use Common Setting for All

Samples:Browse. Click OK

v. Click SET on Multiple Sample Naming window

vi. Type a name in BASE FILE NAME box or locate an existing file

name from BROWSE in “Use Common Setting for All

Samples”. Then click SET

vii. Click SCAN to start scan

6E. Report view

a. To display image select an image analysis software in Scanner Control

window.

b. For multichannel image, channel numbers are displayed in Scanning

Information area of Scanner Control window

The schematic of the results obtained using Typhoon Scanner is shown in Fig 4.

Fig. 4: Schematic representation of images captured Typhoon Imager: Scanning the 2D-DIGE

gel with the help of this scanner, results in three different gel images, one for each dye. A

pseudo-color super-imposed image can also be generated within the software.

6F. Imager shut down

a.       Save all the data with proper name.

b.      Choose FILE:EXIT to close the scanner software.

c.       Shut down the workstation and switch off the power supply of the scanner.

7. Software analysis:

Materials provided: DeCyder 2D Software, Version 6.5.

7A. Image Loader

a. Double click on the DeCyder 2D shortcut icon from the desktop, provide user name and

password to get the access, click on the IMAGE LOADER to begin with.

b. In the image loader window, add gel files to the IMAGES TO IMPORT list by opening

the folder of the scanned images and selecting the desired files.

c. Once all the images are added into the “Images to import” list, do the cropping of gels by

selecting each image and clicking the EDIT GEL option.

d. In the “Edit gel” window, select the required area, crop each image separately and then

save all the images together.

e. In-case file name needs to be changed, click the EDIT option to carry out the desired

change.

f. Create NEW PROJECT in the Center panel of “Image loader” and enter its name and

description. Select the project, click on the highlighted IMPORT button. All files from

“Images to import” get imported into the project folder.

g. Once the image files are imported close the “Image loader” window to start DIA module.

7B. DIA (Differential In-gel Analysis)

a. In the DeCyder 2D Main window, click on DIA option to open the DIA workspace

window.

b. Click on FILE:CREATE NEW WORKSPACE, select a project and all the gel image files

it contains and then click on CREATE to create DIA workspace.

c. In FILE:OPEN WORKSPACE, select the desired DIA workspace file and click OPEN to

open the file for DIA analysis.

d. The gel image gets displayed in the gel view. Now click on PROCESS:PROCESS GEL

IMAGES, select the ALGORITHM, define the ESTIMATED NO. OF SPOTS, and

finally select OK.

e. Now, to set a threshold, select PROCESS: EXCLUDE FILTER, define the values for

each parameter (slope, area, peak height, volume) and click “OK to exclude all spots

within this filter range from the analysis.

f. Individual spots can also be excluded manually from the analysis after taking a proper

look at the 3D view.

g. The spots of interest can be confirmed further by checking for parameters like protein of

interest (POI) and post-translational modifications (PMT) in VIEW: TABLE VIEW

h. Once the user is satisfied with all the spots, the workspace can be saved by clicking

FILE: SAVE WORKSPACE, select or create a new project, where the workspace needs

to be saved and click SAVE

i. Carry out the same steps for other gel images, creating a DIA workspace for each, with

similar parameter settings and save under the project with different DIA file name.

j. Complete all the DIA workspace under the project and close DIA window to start BVA

module.

7C. BVA (Biological Variation Analysis)

a. Click on the BVA icon, to open the BVA workspace window. Now click on FILE:

CREATE NEW WORKSPACE. Under the project option, select all the DIA workspace,

click OPEN to load into BVA workspace.

b. Now define the Experiment by creating folders and assigning the gels into the folder

depending upon the sample type or treatment type. This must be defined in the

EXPERIMENTAL DESIGN VIEW under SPOT MAP MODE view.

c. In the SPOT MAP MODE, the software selects Master gel which represents more

number of spots. User can choose master gel, by selecting the gel of interest and

assigning master tag for it.

d. Once the master is selected, user can go for matching step or can define landmarks so as

to increasing the matching percentage

e. In the MATCH MODE, with the help of gel view in both primary and secondary view,

check for similar spots appearing at the same region across all the gels, mark such spots

as landmark.

f. In the SPOT MAP MODE, click on PROCESS: MATCH to display the following

options:

a. MATCH ALL: matches all gels in the workspace.

b. MATCH SELECTED: matches only the gel selected as primary in the Image

View along with the master.

c. MATCH UNMATCHED AND LANDMARKED :If some gels are matched and

landmarked and some are not, this option matches the pending gels, taking into

account landmarks on the matched gels.

d. OPTIMIZE MATCHING USING WRAPRING: This option wraps the other gel

images to match with master gel and carries out matching.

From the MATCH options select any one and carry out matching process.

g. In MATCH MODE, after matching is done, check for proper matching of spots. In case

of a mis-match, break the match and match the spot manually.

h. To check the matching further, go to VIEW: PROPERTIES: IMAGE VIEW: CHECK

VECTOR VIEW IN MATCH. Vector lines appear on the gel view whose distance and

direction define the matching process. If the vectors are in same direction and are of same

length it implies that the matching is accurate. If this is not the case, try matching the gels

again.

i. Once user is satisfied with matching, the next step is to view spots in PROTEIN MAP.

Here the details of protein expression across all the gels within the project are displayed.

User can employ different statistics tools on the protein spots.

j. For statistical tools click on PROCESS: PROTEIN STATISTICS, select for the type of

test, i.e. paired or Independent. For group to group comparison, options are Average

Ratio and Student T-test and in case of multiple groups select options from 1-way or 2-

way ANOVA.

k. Depending upon the options selected for the “Protein Statistics”, the values for each spot

get calculated and displayed in the PROTEIN TABLE VIEW. To select the significant

spots, user can click on PROCESS: PROTEIN FILTER, define the parameters and set the

threshold value for significance.

l. The filtered protein spots can be later viewed in the APPEARANCE MAP with more

detailed information from the APPEARANCE TABLE VIEW.

m. User can provide the pI and molecular weights of few known spots across the gel and

click on PROCESS: CALCULATE pI AND MOLECULAR WEIGHT to get the values

for other spots.

n. Similarly, the quality of the match can be determined by click on PROCESS:

CALCULATE MATCH QUALITY.

o. Once the significant spots are selected and a list is generated, user can annotate the

selected spots by click on PROCESS: PROPERTIES: IMAGE VIEW: ANNOTATION

OF SPOTS, select any option and provide the input data, which gets annotated for that

spot on the gel.

p. The annotated list can be saved and a print-out can be taken to carry out spot excision on

the selected gel for MS identification.