Withdrawal of Intravenous blood	Sonication of Serum Protein on ice	IgG, Albumin depeltion column packed with slurry	Depletion column	Vacutainer tube and	Acetone precipitated	Dried protein pellet

Whole blood in the	Blood Collection	Centrifugation on whole	Serum separated	Serum	Reconstruction of protein

     QUANTIFICATION OF SERUM SAMPLE

• Prepare the desired quantity of Bradford color reagent.
• Label the tubes as ‘blank’, ‘standards’ and ‘samples’.
• BSA can be taken as the standard. Prepare various dilutions of the BSA standard in different tubes.
• Take 100 µL of the sample and dilute to 500 µL using rehydration buffer (100 µL of sample + 400 µL rehydration buffer) and mix well.(Dilution is optional, Proceed with dilution depending upon sample availability).
• Take 500 µL of standard and samples in their respective labeled tubes.
• Add 500 µL of Bradford color reagent to all the tubes including blank and standards.
• Incubate the tubes at room temperature for 10 minutes.
• Then measure the optical density of each tube using a spectrophotometer at 595 nm.
• Subtract the O.D of the blank from all other readings to obtain the actual OD.
• Plot a linear standard curve of O.D of the standards against their respective concentrations.
• Plot the OD of the unknown samples and then extrapolate to the concentration axis to determine the unknown concentration.

Label tubes for assay	Add the sample to respective tube	Addition of Bradford	Color development after addition of dye	Read the O.D on Spectrophotometer

     ISOELECTRIC FOCUSING

        Rehydration of IPG strip:

           Materials required:

• IPG strips-24 cm 4-7 pl.
• Rehydration solution- 8M urea, CHAPS (2% w/v),BPB (0.002%), IPG Buffer 0.5%(v/v).
• IPG buffer and DTT (should be added just before use).
• 5 µL IPG buffer [pI 4-7] and 6.2 mg of DTT was added to an aliquot of 1 mL of rehydration solution.

          Method

• Level the re-swelling tray.
• Thaw the tube containing the proteins on ice and then centrifuge at 1000g for 1 minute at oC.
• A maximum of 1.2 mg of protein and total of 450 µL protein containing solution can be loaded on each strip. Therefore, calculate the volume of suspension accordingly and add the appropriate volume of both sample and rehydration buffer into a fresh tube. [if your sample volume is 90µL then; 360 µL rehydration solutions+ 90µL protein solution (from the upper layer of the tubes) = Total 450µL].
• Vortex, give a brief spin for settling debris if any.
• Add the sample into the tray as a streak.
• Place the strips in the channels with the gel side facing down and the ‘+’ sign on the strip placed against the sloped end of the channel.
• Allow strips to rehydrate with protein for 30 minutes.
• Then cover each strip with 3 mL of cover fluid (mineral oil). Also, place 2 mL of mineral oil in adjacent channels which do not have the strip.
• Carry out passive rehydration of gel for 12-14 hrs.

        Iso-electric focusing-IEF

• Clean the IEF tray with non-ionic detergent. Wipe with dust free paper.Check the level of the IEF tray to ensure that it is on a flat, smooth surface.
• Place the rehydrated strips carefully in the IEF tray of IEF machine with the gel side facing upwards and the ‘+’ sign towards the top, as indicated on the tray. (please match the that positive end of the strip matches with that of negative end of machine’s electrode).
• Cover the strip with mineral oil in each lane. Place paper wicks at both end of the strips, then mount the electrodes and start the focusing.Following example of protocol which can be used for IEF.

After IEF, the strips can be stored at -200C in trays or can be used immediately for separation in the 2nd dimension.

IEF apparatus	Rehydration of strip with protein sample	Addition of mineral oil	IEF Instrument	IEF Instrument with IEF tray

Placing wick on	Mounting electrodes	Mounting electrodes	Adding of mileral oil	IEF settings ready for focusing

      SECOND DIMENSION SEPARATION-SDS PAGE

       Gel casting [12.5% SDS-PAGE]

• Clean the glass plates and casting system and dry them well.
• Mix the following components and make up the solution to 200 mL  with distilled water to prepare a 12.5% gel mix, which is sufficient for two gels:

• Once the gel mix is ready, APS and TEMED are added just before pouring the gels and mixed thoroughly.
• Pour the gel solution in between the glass plates through the filling channel.
• Immediately after pouring, spray a 0.1% SDS solution on top of the gel surface.
• Then allow the gels to polymerize for an hour.

       Equilibration of the IPG strips

• Remove the IPG strips from -20oC storage unit and allow them to thaw for 5 minutes at RT.
•  Place the strip for 15 minutes in the rehydration box well containing the equilibration buffer 1 containing DTT [detailed composition provided in the annexure]. Provide gentle shaking on a horizontal shaker.
•   Next, transfer the strip to the second rehydration box containing the equilibration solution 2 containing  iodoacetamide and subject it to gentle shaking for 15 minutes.

       Placement of the strips on SDS Gel surface

• Wash the strip gently after the 2nd equilibration [10 sec] with 1X electrophoresis buffer. [Keep the strip in rehydration tray and flood with buffer].
• Then, place the strip carefully on the gel surface, constantly ensuring that there is no air bubble being trapped.
• The strip side having the ‘+’ sign must face the left.
• Next, pour around 2.5 mL of the lukewarm overlay agarose solution on top of the strips and allow it to solidify for 1 minute. The gel is now ready for electrophoresis.
• Place the ready gels into the electrophoresis tank containing the appropriate buffer, while ensuring that the buffer level matches the mark.
• Run the set up at 100 V for an hour followed by 350 V.
• After completion of the run, carefully disassemble the plates and remove the gel for staining.
  

Small gel assembly	Small gel casting	Small gel assembly	Assembly of appartus	Glass plate assembly

Glass plate	Glass plate assembly	Mounting of molecular weight marker	Buffer tank	Serum proteins separated on 2DE stained with coomassie

      STAINING AND DESTAINING

 

• Once electrophoresis is complete, wash the gel thoroughly with distilled water to remove any bound SDS by placing the gel in tray containing distilled water for 5-10 min with intermittent changing of water.
• Then place the gel in a staining solution composed of Coomassie Brilliant Blue for around 5-6 hours with gentle rocking on a mechanical shaker.
• Once staining is complete, transfer the gel to a destaining solution [composition provided in annexure] for 5-6 hours with gentle rocking.
• Finally, wash the destained gel twice with distilled water before scanning.
• Carefully place the gel on the imaging platform, capture the image and save it with an appropriate filename.
• Carry out analysis using the software to compare the proteome profiles obtained for the disease & control.

Partly stained gel	Destaining process	Destaining process	Imaging of the gel with scanner	Serum proteins separated on 2DE stained with coomassie	Silver stainning of serum protein

      DATA ANALYSIS

It is essential to a analyze the 2-DE gels in order to draw any conclusion about proteome level changes. Analysis is mostly done with the help of software which enables simultaneous comparison of enormous number of spots across various gels. Gels with proteins of various biological conditions such as diseased and healthy, drug treated and untreated can be analyzed to draw a conclusion about unique or differentially  expressed proteins. Various links are provided will guide you over the analysis process. These files can be downloaded for use.

A)    We have demonstrated the process of analyzing gel using a simulation. This simulation guides you to get a real tome feel of data analysis pattern. You may click HERE to get an access to the simulator. Following are details of the simulation;

• Make match set: This option creates a match between gels. After option is enabled the window displays similar spots over 2 gels. Now one may proceed with getting further information on spots.

• Spot ID: Tracking each spots in the gel on the course of analysis iS made easy be designating each spot with a spot ID. For knowing spot ID of a particular spot, please click on the desired spot and press the button of spot ID. This displays the spot ID of clicked spot and also the corresponding spot in matching gel.

• Molecular weight & pH: Molecular weight & pH of the spot can be displayed by clicking on a spot and followed by clicking the molecular weight option.

• Spot intensity: This is a quantitative measurement of  protein across the spots. This is a relative figure value obtained can be further used for statistical analysis.

• Intensity comparison: This is a graphical representation of differences in the intensities. To enable the option, click on the desired spot then click on the option.

• 3-D view: Each spot on the gel can be visualized in a 3D manner. This option displays the intensity of spot in as a peak. Smoothness of the peak depicts the goodness of the spot. This also helps for differentiate between false and original spots.

• Fold change: This option helps the user to conclude on whether or not the spot is differentially is expressed. The fold change number indicates the changes in the protein expression level across the two different biological conditions. Option 9 will display all three options of Intensity comparison 3-D view and Fold change simultaneously.

Practicing on the simulation helps for getting familiarized on the analysis process. In the next stage one may proceed using the commercially available softwares for analysis.

 B)  Out put of all three experiments are (3 gel pairs, 2 different biological conditions) available for download in the DOWNLOAD section of the website.

 C) Various companies provide analysis softwares on a trial use basis. One may download these software for practicing the analysis of provided 2-DE gel images.

 D) You may click to get demonstration on analysis:-Analysis Demonstration.ppt . Analysis is done using one of the commercially available softwares. The power point presentation guides in all the steps right from uploading of the gel till getting the final out put of differential expression.

Analysis Demo- 1	Analysis Demo- 2	Analysis Demo- 3	Analysis Demo- 4	Analysis Demo- 5	Analysis Demo- 6	Analysis Demo- 7

Analysis Demo-8	Analysis Demo-9	Analysis Demo-10	Analysis Demo-11	Analysis Demo-12	Analysis Demo-13	Analysis Demo-14

Analysis Demo-15	Analysis Demo-16	Analysis Demo-17	Analysis Demo-18	Analysis Demo-19	Analysis Demo-20	Analysis Demo-21

Results: Comparison of control and stress-induced gels will reveal those proteins that are expressed in the presence and absence of the stress condition. Also, differential expression between the gels can be studied by comparing intensities of various spots thereby knowing which proteins get up-regulated or down-regulated. In addition to comparing case and control, a match set can be created for all the gels which will provide an understanding into number of spots that are different across the gels, by what percentage they vary in other parameters like intensity, size etc. The spots of interest can be excised from the gel, digested and then analyzed by mass spectrometry techniques such as MALDI-TOF-MS. Details of the experimental procedures will be provided in second module.