Fig. 1: Experimental design for 2-D gel electrophoresis: Cultured cells are grown under

Transcription

1 Fig. 1: Experimental design for 2-D gel electrophoresis: Cultured cells are grown under stringent conditions. The cells are harvested at mid-log phase, proteins extracted from whole cell lyzates and the protein concentration is determined. IPG strips are then rehydrated with sample

2 and separation is carried out by IEF. The equilibration step prepares the strip for SDS-PAGE. Once the proteins are separated completely, the gels are scanned and analyzed using software tools that identify global proteome pattern of the breast cancer cell line. Protocol: 1. Culturing of breast cancer cell line: Materials provided: Breast cancer cell line. Materials required: Autoclaved MilliQ water,tips and glassware, gloves, two 75cm 2 cell culture flasks, cell scraper. Reagents: Ethanol, minimum essential medium, fetal bovine serum, sodium bicarbonate, antibiotic solution, insulin, trypan blue, PBS buffer. Instruments Required: Laminar Air-flow, CO 2 incubator, microscope, hemocytometer, centrifuge, Vibro cell sonicator. a) Autoclave the water, glassware and tips required at 15 lb pressure and 121 C for 15 min. b) Prepare the Miminal Essential Medium as per manufacturer s instructions using the autoclaved water. Filter the medium using a 0.22 micron filter paper into a 1L glass bottle. c) Clean the working bench of laminar air flow with ethanol and turn on the UV lamp for 15min, after which it is switched off.

3 d) Wear fresh gloves and place all the materials required on the working bench, along with a culture flask containing breast cancer cells. e) Prepare complete media by adding 10% fetal bovine serum, 2.2 g/l sodium bicarbonate, 1% antibacterial and antimycotic solution containing streptomycin, amphotericin B and penicillin and 10 g/ml of human insulin to the Mimimal Essential Medium. f) Wash the cells in the culture flask with PBS twice and then scrape the cells off the flask with the help of a cell scraper. Collect the resulting solution in a clean tube and centrifuge at 800 x g for 10 min at room temperature to get the cell pellet. g) Resuspend this pellet in 1 ml of complete media and count the number of cells by the trypan blue exclusion method, using a hemocytometer. h) Aliquot the required amount of cell suspension to two 75cm 2 cell culture flasks containing 7 ml of complete media and place the flasks in a CO 2 incubator set at 37 C, 5% CO 2 and 95% humidity. Fig. 2: 75cm 2 cell culture flasks containing the breast cancer cell line cells in 7 ml of complete media.

4 i) Allow the cells to grow till they reach around 70% confluency. Fig. 3: Microscopic image of breast cancer cell line, growing on a 75cm 2 cell culture flask. j) Harvest the cells in PBS using a cell scraper as before and centrifuge the solution at 800 x g for 10 min at room temperature to get the cell pellet. k) The cell pellet obtained can either be used immediately for protein extraction or stored at -20 C for later use. 2. Sample preparation: Materials provided: Trizol reagent. Materials required: Autoclaved 1.5 ml eppendorf tubes and pipette tips. Reagents: Acetone, chloroform, ethanol, lysis buffer and distilled water.

5 Instruments Required: centrifuge, freezer. a) Harvest the cells in PBS using a cell scraper as before and centrifuge the solution at 800 x g for 10 min at room temperature to get the cell pellet. b) Wash the cell pellet twice with PBS c) Resuspend the pellet in lysis buffer containing 50 mm tris-hcl, 150 mm NaCl, 0.1% triton X-100, 4 mm EDTA, 50 µg/ml PMSF, 50 mm NaF, 0.2% NP-40, 1 mm DTT and protease inhibitor cocktail. d) Kept the tube on ice for 1 h with intermittent vortexing. e) To lyse the cells completely, sonicate the cells using Vibro Cell sonicator with following settings: 4 cycles of 1 sec pulse with 2 sec gap in between, at 30% amplitude. Fig. 4: Cell lysis by sonication method using a Vibro Cell Sonicator.

6 f) Centrifuge the resulting solution at 12,000 x g for 15 min at 4 C to remove the cell debris. Collect the clear supernatant in a separate tube. g) Add 1 ml trizol reagent to this supernatent. h) Immediately add 200 ul chloroform to the same mixture, shake vigorously for 15 sec & incubate for 15 min at RT. i) Centrifuge at 12,000 g for 15 min. Cell sample gets separated into three distinct layers. Fig. 5: Whole cell lysate is separated into three distinct layers during protein isolation by Trizol method. j) Carefully remove upper transparent layer containing RNA using a micropipette. k) 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. l) Separate the supernatant containing protein collect into a fresh tube. Retain and store the DNA pellet at 20 C.

7 m) 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. n) After incubation centrifuge at 12,000 x g for 5 min. o) Discard the supernatant, retain the pellet of protein. p) Wash the protein pellet with 95% (95% ethanol+5% water) ethanol or acetone (4 times). q) After each wash give a brief spin to settle proteins, discarding the supernatant each time. r) Dry the pellet in the room temperature. s) Reconstitute the dried pellet in lysis buffer and store the protein solution at 20 C till further use. 3. Protein Quantitation: The modified Bradford method used for protein quantification is explained below. Materials provided: Bradford reagent. Materials required: Sample, pipette, eppendorf tubes, ice, and tips. Reagents: BSA and distilled water. Instruments Required: Mini Centrifuge, bench top vortex and spectrophotometer. a) Label the tubes as standard (S1 to S10), blank (B1, B2) and sample (T1 to T10). b) Pipette out Bovine Serum Albumin (BSA) in increasing order to get the desired range of standard protein concentration.

8 c) Pipette out required amount for blank,i.e. distilled water, and samples into the labeled tubes. Thaw the samples completely before use and if required dilute the samples. d) Makeup the final volume to 1ml by adding distilled water. (In some case sample buffer can be used instead of distilled water) e) Add equal amount of Bradford reagent to the tubes. f) Mix the solution in the tube by vortexing followed by incubation for 10 minutes in the dark at room temperature. Fig. 6: In the presence of proteins, the Coomassie Brilliant Blue G-250 dye forms a stable complex that is blue in colour. Intensity of the colour is directly proportional to the concentration of the protein in the solution. g) Turn on the spectrophotometer and set it at 595nm. h) Clean the cuvette with distilled water, transfer blank solution into the cuvette and set the instrument to zero. i) One by one, transfer the standard and sample solutions to the cuvette and make a note of the absorbance reading.

9 j) Plot a graph of standard concentrations (on X-axis) against the absorbance reading (on Y- axis). Fig. 7: Graph showing the standard curve while determining concentration of protein by Bradford method. k) Find the sample protein concentration by using the corresponding absorbance reading and extrapolating the graph. l) Note: In case the absorbance reading of the sample is high, try diluting the sample and taking the reading once again or increase the range of the standard concentration used. 4. First Dimensional - Iso-Electric Focusing (IEF): Materials provided: IPG strips, IPG buffer and IPG phor instrument. Materials required: Sample, ice, lint free tissue, wicks, forceps, tips, reswelling tray and manifold.

10 Reagents: Urea, thiourea, CHAPS, SDS, DTT, BPB, cover fluid. Instrument required: mini Centrifuge, horizontal shaker, pipette bench top Vortex and IPGphor. 4A. Rehydration of IPG strips: a. Prepare 25 ml of rehydration solution, containing 7 M urea, 2 M thiourea, 4% CHAPS and 0.001% BPB. Mix properly and store at 20 C. b. Before use add 2% Pharmalyte or IPG Buffer and 40 mm DTT. Mix thoroughly by vortexing. c. Depending upon the length of the strip used, pipette out the amount of sample required and make up the final volume with rehydration solution. Mix the solutions in the tube by vortexing and keep it on ice bath. Fig. 8: Solutions containing the sample, rehydration buffer IPG buffer and DTT are mixed thoroughly by vortexing on a vortex mixer.

11 d. Clean rehydration tray with lint free tissue. To a dry lane add the above sample solution. Fig. 9: Image of the reswell tray with the lid slightly drawn out. e. Take out the IPG strip from 20 C freezer and keep at room temperature for 5minutes. Take out the packing material and remove the protective cover with forceps. Fig. 10: Commercially available IPG strips are shown. Each strip is placed in a separate lane and sealed independently.

12 f. Place the IPG strip with gel side facing down, carefully placing the strip in the lane to make contact with the solution. Try to avoid air bubbles in-between the solution and strip. Fig.11: IPG strip is placed gel-side down in a lane of the reswell tray that contains the sample solution. g. Cover the reswelling tray with the lid and do not disturb the set-up till 20-30min. h. After 20-30min pipette out around 2ml of cover fluid in the lane over the strip to avoid drying of the strip due to evaporation. Fig. 12: The lane containing the IPG strip placed over the sample solution is filled with Cover fluid to prevent gel drying and sample precipitation.

13 i. Place the set-up at room temperature and do not disturb the set-up till 15-18hours. 4B. Focusing of IPG strips: j. Take out the manifold, clean it with lint free tissue, open the lid of the IPGphor unit and place the manifold over the unit in the groves provide. Balance the unit properly with the aid of a spirit level. Fig. 13: The lid of the IPGphore unit is lifted and the manifold is placed in the groves provided for this purpose. k. After the rehydration step is completed, remove the lid of reswelling tray and take out the strip with forceps and tap it on to the tissue to remove excess cover fluid. l. Place the strip in the channel of manifold with gel side up; making sure that the positive end of strip is placed on the positive side of the unit. Position the strip according to the length marked on the unit.

14 Fig. 14: Rehydrated IPG strips are placed on the manifold of the IPGphore unit with the help of forceps. m. Cut the wicks depending upon the number of strips used for focusing. Two wicks are used per strip, one on each end. Wet the wicks with 150ul of distilled water and with forceps, place wicks over the gel end of the strips. Fig. 15: Wet paper wicks are placed on the ends of the IPG strips placed on the manifold before the start of the IEF run. n. Fill the manifold with fresh cover fluid in all the channels.

15 Fig. 16: Cover fluid is added to all channels of the manifold to prevent the gel drying and sample precipitation. o. Place the electrodes on top of the wicks to make contact. Once both the electrodes are placed on either end of the strip, lock the electrode assembly. Fig. 17: Electrodes are positioned over the paper wicks which are placed at both the ends of the IPG strips. p. Close the lid of the unit and turn on the instrument. Wait for the unit calibration and later click open the shortcut for IPGphor unit from the desktop to open the software window.

16 q. In the software window set the parameters, like number of strips, details of the strips, etc. Set the protocol for focusing by defining the voltage and time required for each step. Fig. 18: Schematic representation of IPGphore software window showing all the parameters which need to be defined before starting the IEF run. r. Once the parameters are defined, connect the instrument with the software; transfer the protocol to the unit. Click on PLAY/RUN. s. Monitor the focusing step, in case the voltage does not achieve the set value, STOP the run. Open the lid of unit, remove the electrode and check for wick color. If yellow, change the wicks with fresh ones and continue the focusing.

17 Fig.19: Images of the IPGphore software window during an IEF run. A) Blue line indicates the voltage attained by the unit during the run while red line indicates the defined voltage ramp-up. Since the actual voltage attained by the unit does not match the defined voltage ramp-up, the run has to be paused and the wicks need to be changed. B) Example of a good IEF run wherein the actual voltage attained by the unit matchs the defined voltage ramp-up. t. After the focusing step, switch off the unit, open the lid of unit, remove the electrodes and wicks and with forceps take out the strips. The strips can be stored at 20 C for future use or can be taken for equilibration step immediately. 4C. Equilibration of strips: a. Prepare 20 ml of Equilibration buffer, containing 6 M urea, 75 mm Tris-HCl ph 8.8, 29.3% glycerol, 2% SDS and 0.002% bromophenol blue. Make up the volume with distilled water, mix by vortexing and can be store at 20 C for later use. b. From this 20ml take out 10ml to prepare Equilibration buffer- I by adding 10 mg of DTT. Mix properly by vortexing before use.

18 c. Place the focused strip in a clean lane of a tray, add Equilibration buffer- I and place the tray on the horizontal shaker for 15min. Fig. 20: IPG strip is placed in Equilibration buffer I containing DTT to break the disulfide bonds. The strip is allowed to remain in the solution for 15min on a shaker. d. Meanwhile, for the other 10ml of buffer, add 25mg of iodoacetamide to prepare Equilibration buffer- II. Mix properly by vortexing. e. After 15min, lift up the strip with the help of forceps, tap one end onto a tissue paper to remove remaining buffer-i solution, and place it in new lane of the tray. f. Add Equilibration buffer- II to this lane and place the tray on the shaker for 15min. g. After 15min, lift up the strip with the help of forceps and tap one end onto a tissue paper to remove remaining buffer-ii solution h. Rinse the strip with running buffer solution to prepare it for second dimension electrophoresis.

19 Fig. 21: IPG strip is washed in the running buffer solution before loading onto the SDS- PAGE. 5. Second dimension - SDS-PAGE: Material Provided: Glass plates and plastic sheets. Material Required: Focused and equilibrated IPG strips Reagents: Acrylamide-bis-Acrylamide solution, Distilled water, Tris-HCl, SDS, APS, TEMED Instrument required: Gel casting unit, Ettan DALT running unit, power pack, oven and magnetic stirrer. 5A. Preparation of the solutions. a. Laemmli buffer: 250 mm Tris base, 1.92 M glycine, 1% SDS to total volume of 10 ml in distilled water. Mix the solution thoroughly using magnetic stirrer. b. SDS buffer: 10% SDS in a total volume of 50 ml.

20 c. Sealing Agarose: 25 mm Tris base, 192 mm glycine, 0.1% SDS, 0.5% agarose, 0.002% bromophenol blue to a total volume of 100 ml in distilled water. d. Acrylamide cocktail: Mix Acrylamide Bis-Acrylamide solution (29:1 ratio), 1.5 M Tris- HCl(pH 8.8), SDS (10%), APS (10%) and TEMED to the required gel percentage and required volume with distilled water. 5B. Gel Casting. a. Prepare the gel casting unit, by placing the glass plates next to each other with plastic sheets filling the space in-between. Try casting an extra gel as reserve and include dummy plates if required. b. Check the casting unit for any leakage by filling it with distilled water. Keep the set-up ready before preparing the acrylamide cocktail. Fig. 22: The gel casting unit is checked for leakage by filling it with distilled water. c. Prepare acrylamide cocktail, containing all the ingredients except APS and TEMED, and mix it properly. Just before pouring the cocktail, add freshly prepared APS and TEMED. Mix it thoroughly. Now slowly pour the cocktail into the gel casting unit through the

21 filling channel and try to avoid the formation of air bubbles. Leave some space on top of the gel between the glass plates for placing the focused IPG strips. Fig. 23: The acrylamide cocktail is poured into the gel casting unit and the gel is allowed to polymerize between the glass plates. d. Now quickly spray SDS solution on top of the gel casting unit so that the cocktail is not exposed to air and a uniform surface is maintained for placing the strip.

22 Fig. 24: The plates in the gel casting unit are overlayed with 0.1% SDS solution to prevent exposure of the setting to air. e. The gel casting unit must not be disturbed and must be placed on leveled platform. Around minutes are required for the gel to polymerize. f. Disassemble the casting unit, take out each gel plate, decant the excess SDS buffer from top of the gel, place it on the gel stand and pipette out distilled water over the gel and make it ready for running the second dimension. 5C. Gel Run. a. Melt the sealing agarose in oven and keep it for cooling so that it attains optimum temperature for sealing. b. Lift the focused and equilibrated IPG strip with forceps and carefully place it on the SDS- PAGE gel, ensuring proper and uniform contact with the gel and trying not to disturb the gel coat. Fig. 25: Focused IPG strips are placed on top of the gels cast between the glass plates.

23 c. Decant the water on gel carefully by tilting the glass plates and remove any remaining water with the help of absorbent tissue paper.now pipette out sealing agarose from one end. Make sure the contact between the strip and gel is uniform without any air bubbles in-between. Let the sealing agarose solidify. Fig. 26: Sealing agarose solution is poured over the focused IPG strips, placed on top of the SDS-PAGE gels. d. Prepare the running unit by pouring the Laemmli buffer, make the proper dilution for lower chamber buffer (1X) and upper chamber buffer (2X). Fig. 25: 1X running buffer is filled in the lower tank of the Electrophoresis unit. e. Now place the gel plates into the groves of the running unit, keeping them on either side to make a balance. Make-up for the remaining groves with dummy plates.

24 Fig. 26: A) Gel plates are placed on both sides of the running unit. B) The running unit is then placed in thelower buffer tank of the Electrophoresis unit. f. Place the upper chamber tightly on top of the glass plates. Now pour the upper chamber buffer (2X). Check for any leakage. Fill both the chambers with the respective buffers atleast upto the minimum level mark on the unit. Fig. 27: The upper chamber is placed tightly on top of the running unit, and the upper tank buffer is then filled. g. Close the lid of the running unit, make proper connections to the power pack. Apply 5watts/gel for initial 1 hour and apply 15-17watts/gel for run to complete.

25 Fig. 27: The lid of the running unit is connected to the power pack. h. Power pack can be switched off when the marker dye front runs out from the other end of the vertical gel. i. Carefully disassemble the plates and remove the gels for staining. 6. Staining and Scanning of 2-D electrophoresis gels: Material Provided: Glass Tray. Material Required: Gel cutter, gloves Reagents: Coomassie Brilliant Blue R-250 dye, Methanol, Acetic acid and distilled water Instrument required: Horizontal Shaker, Gel scanner (Lab Scan) 6A. Preparation of the solutions.

26 e. Staining solution: 50% (v/v) methanol, 0.05% (w/v) Coomassie brilliant blue R-250, 10% (v/v) acetic acid and 40% H 2 O f. Destaining solution: 50% (v/v) methanol, 10% (v/v) acetic acid and 40% H 2 O 6B. Staining and destaining the gels. g. After electrophoresis, remove the gels carefully from the glass plates into glass trays and wash them thoroughly with distilled water for 5-10 minutes to remove any bound SDS. h. Decant the water and pour the staining solution over the gels in the glass trays. Leave the trays on a horizontal shake for 5-6 hours with gentle rocking. Fig. 28: The 2-D electrophoresis gel is placed in a tray containing the Coomassie Blue staining solution and is gently rocked on a shaker for 5-6 hours. i. Then decant the staining solution. Pour the destaining solution over the gels and leave the glass trays on the shaker for a further 5-6 hours with gentle rocking.

27 Fig. 29: The stained 2-D electrophoresis gel is placed in a tray containing the destaining solution and is gently rocked on a shaker for 5-6 hours. j. Wash the destained gel with distilled water twice with water, leaving the tray on the shaker for 1-2 hours each time. 6C. Scanning the gels. a. Turn on the Scanner Unit. Open the ImageScanner III LabScan 6.0 software and choose the setting for the scanner. Carry out scanner calibrations if required. b. Open the transparency unit and wipe the surfaces with lint-free tissue. c. Place the gel on the scanner, taking care that the gel does not break and that air-bubbles do not get trapped between the gel and the scanner surface.

28 Fig. 30: Stained 2-D electrophoresis gel is carefully placed on the scanner. d. Preview the image to be scanned and define the zone of interest using the Crop tool. e. Carry out the final image scan by clicking on the Scan icon in the toolbar. f. Adjust the orientation of the gel image and the contrast of the image to be able to view all the spots present on the original gel. Fig. 31: Image of a typical 2-D electrophoresis gel showing breast cancer cell line proteins separated on a 4-7 ph range on the X axis and molecular weight on the Y axis

29 g. Save the scanned image as both.tiff and.mel files. h. Remove the gel from the scanner and wipe the surface of the scanner with clean lint-free paper. 7. Image analysis: Material Provided: ImageMaster 2D Platinum 7.0 software. Material Required: Gel images Instrument required: Computer system having Microsoft Windown XP or Vista operating systems and atleast 500 MB of RAM. a) Create a master folder and import all the gel images to be analyzed into it. Open the images with the help of the software and label them appropriately. Fig. 32: Schematic representation of the software display showing various tools.

30 b) Create a project in the workspace and import different gel images into a Match folder. Fig. 33: Create a project in the software c) Crop the gel images to exclude the regions without spots from the analysis. d) Define the spot detection parameters before using the automated spot detection tool of the software. Then manually edit the spots, using the Edit Enable tool in the Edit: Spots menu. This is an important step and care should be taken not to select a wrong spot or miss an important protein spot. Alternatively, the spots on the gel image can be detected manually by zooming into the gel image and drawing the outline of each spot.

31 Fig. 34: Parameters for spot detection are defined and the spots are detected either manually or by using the automated spot detection tool. e) Each spot is designated a Spot ID, which can be seen by clicking on the desired spot and selecting the button for Spot ID. This helps in tracking each spot in the gel during the course of analysis and also in locating the corresponding spot in matching gel. f) If pi/mw standards have been run on the gel along with the sample, then clicking on the spot of interest and selecting the pi_mw option can give the relevant information. g) Selecting Spot Table in the Reports menu gives the spot intensity for each spot, along with the spot ID and the calculated volume, pi and MW values. The spot intensity is a relative measure of proteins across all the spots, and can be used further for statistical analysis.

32 Fig. 35: Representation of the spot analysis table. h) Spots can also be matched across all gel images present in the Match folder by using the option of Match Gels in the Edit:Matches menu. This process displays the number of spots that are matched and unmatched across the gels. Fig. 36: Gel images are matched to locate the common and/or unique in the two gels. i) Statistical parameters of spot matching across different gels, such as coefficient of variation, standard deviation, t-test values, etc. can be obtained from the Gel Analysis Table. This can be displayed by selecting Reports: Analyse Gels: Table from the toolbar. j) The extent of matching can also be seen with a 3-D graphical representation of the spots.

33 Fig. 36: 3D view of the spots on the two gels. Fig. 37: A schematic representation of all the processes involved in global expression analysis, starting from preparation of protein sample to analysis of 2-D gels is shown.

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