Wendel Lab Isoelectric Focusing of Total Proteins Protocol
Department of Ecology, Evolution, and Organismal Biology
Iowa State University, Ames, IA 50011, USA
Materials and Reagents
1. Protean IEF Cell, Protean IEF system 24 cm disposable trays ( catalog number, 1654043), and Isoelectric point gel (IPG) focusing tray are from Bio-Rad ( Bio-Rad Laboratories, Hercules, CA, USA ).
2. Linear IPG strips (pH 4-7, 24 cm; GE Healthcare, Piscataway, NJ, USA). (Note: It is important to use the narrow-range IPG strip to avoid spot overlap to obtain distinct protein spots).
3. IPG buffer ( pH 4-7, 24 cm; GE Healthcare, Piscataway, NJ, USA).
1. Add 1 mg protein to a 1.5 mL microfuge tube and bring the final volume to 450 µL with IEF extraction buffer.
2. Add 2.25 µL (final concentration 0.5%) of the correct IPG buffer to the same tube.
3. Mix the sample by vortexing and centrifuge at 14000 rpm for 5 min to remove insoluble materials.
4. Pipette the supernatant into the IPG focusing tray starting from one end to another end. (Note: Care must be taken to avoid introducing any bubbles ).
5. Peel apart a dehydrated IPG strip (pH 4-7; 24 cm) using forceps and place the dried acrylamide side face down into the sample well in the IPG focusing tray. (Note: Remove bubbles if trapped by lightly tapping the upper surface of the IPG strip. Laying the IPG strip down from one side to the other gradually should prevent bubble formation ).
6. Cover the focusing tray with the lid and keep inside a plastic bag to minimize evaporation.
7. Allow the IPG strip to rehydrate for 90 min at RT.
8. After rehydration is complete, wet two paper wicks (catalog #165-4071) with deionized water (10 μl per paper wick). Carefully lift the ends of each IPG strip with forceps and insert a wet paper wick between the IPG strip and the electrodes.
Wick papers can function as ion traps, sequestering ionic components away from the IPG gel strip.
9. Overlay the IPG strip with mineral oil (approximately 2.5 mL) to prevent dehydration. (Note:Mineral oil should be applied drop-wise from the center of the IPG strip until the entire strip is covered).
10. Place the IPG focusing tray into Protean IEF cell unit.
11. Perform active rehydration (10 hour at 50 Volt), followed by three-step focusing protocol: 100 V for 100 V-hour, 500 V for 500 V-hour, and 8000 V for 99 kV-hour (program 24 cm).
Materials and Reagents
1. Ettan DALTtwelve electrophoresis unit (GE Healthcare, Piscataway, NJ, USA).
2. Acrylamide stock (30.8% T): 30% (w/v) acrylamide and 0.8% N,N’-methylene bis-acrylamide. Filter sterilize and store at 4-8ºC. (Note: Unpolymerized acrylamide is a neurotoxin, so care should be taken to avoid exposure).
3. 1.5 M Tris-HCl, pH 8.8. Filter sterilize and store at 4-8ºC.
1.5M × 121.1g/mol × 500mL = 90.825g
4. 0.5 M Tris-HCl, pH 6.8. Filter sterilize and store at 4-8ºC.
0.5M × 121.1g/mol × 500mL = 30.275g
5. 10% (w/v) SDS in deionized water. Filter sterilize and store at RT.
6. 10% (w/v) ammonium persulfate in deionized water. Use freshly prepared solution.
7. Fresh TEMED (N,N,N’,N’-tetramethyl-ethylenediamine; product number, BP150-20) is important for polymerization of gel. (Note: The quality of TEMED may decline with time after opening, and therefore gels take a longer time to polymerize. It is recommended to buy a small bottle of TEMED and to store at 4ºC).
8. Displacement solution: 375 mM Tris-HCl, pH 8.8, 50% (v/v) glycerol, and trace of bromophenol blue in deionized water. Store at 4-8ºC.
Tris-HCl 1.5M, pH8.8 375mM × 500mL /1.5M= 125mL
9. PeppermintStick phosphoprotein molecular weight standards (Invitrogen, Carlsbad, CA, USA; product number, P33350). Store in aliquots at -20ºC. (Note: PeppermintStick molecular weight standards contain β-galactosidase [116.25 kD]), bovine serum albumin [66.2 kDa], ovalbumin [45.0 kDa], bovine β–casein [23.6 kD]), avidin [18.0 kDa] and lysozyme [14.4 kDa]).
10. SDS equilibration buffer: 50 mM Tris-HCl, pH 8.8, 6 M (w/v) urea, 30% (v/v) glycerol, and 4% (w/v) SDS. Filter-sterilize and store in single-use aliquots at -20ºC in 15 mL Falcon tubes.
11. Reduction solution: 2% (w/v) DTT in SDS equilibration buffer. Prepare right before use.
12. Alkylation solution: 2.5% (w/v) iodoacetamide in SDS equilibration buffer. Prepare fresh and protect from light.
13. Electrode wicks ( Protean IEF system; Bio-Rad Laboratories, Hercules, CA, USA; catalog number, 1654071 ).
14. Agarose sealing solution: Add 125 mg (0.6%) agarose to 20 ml of 1 x SDS running buffer plus a few grains of bromophenol blue.
16. Rocking platform (Rocker II; Boekel Scientific, Feasterville, PA, USA, Model 260350).
Assembling the Ettan DALTtwelve Gel Caster Unit
1. Tilt the DALTtwelve unit back so that it rests on its support legs. (Note: We recommend reading the user manual for Ettan DALTtwelve electrophoresis system ).
2. Place a thicker separator sheet against the back wall to easily remove the last cassette from the gel caster unit after polymerization.
3. Clean each glass plate carefully with deionized water and ethanol using Kimwipes.
4. Fill the caster by alternating cassettes with separator sheets. End with a separator sheet, and then use the thicker separator sheets to bring the level of the stack even with the edge of the caster.
5. Lubricate the foam gasket with a small amount of GelSeal and place it in the groove on the faceplate.
6. Turn four black-knobbed screws into the four threaded holes across the bottom until they are well engaged. Usually two to three full turns are enough.
7. Place the faceplate carefully onto the caster with the bottom slots resting on their respective screws. Screw the four remaining black-knobbed screws into the holes at the sides of the faceplate and tighten all eight evenly. Assembled unit is now ready to cast gels. (Note: Make sure the sealing gasket is compressed evenly by the faceplate and forms a tight seal with the caster. Do not over-tighten the screws.)
1. To cast six gels, add 150 mL of acrylamide stock, 94 mL of 1.5 M Tris-HCl, pH 8.8, 3.75 mL of 10% SDS, and 126 mL of deionized water in one liter sidearm flask. This is separating gel solution.
2. Place the flask on stir plate, add a medium size stirring bar, and stir the solution.
3. Connect sidearm to vacuum, cover top opening with solid rubber stopper, and apply vacuum for 30 min.
4. Turn off vacuum, remove rubber stopper, and disconnect hose.
5. Add 1.8 mL of 10% ammonium persulfate while stirring the solution.
6. Add 120 µL TEMED, and continue to stir for 30 sec. Move quickly to cast the gels.
7. Slowly pour the gel solution into the caster through the hydrostatic balance chamber until it is about 2 cm below the desired gel height.
8. Pour the displacement solution into the chamber until it is 0.25 cm below the surface of glass plates, and then immediately place the feed tube into the grommet to stop the flow (Note: The flow of displacement solution can also be stopped by clamping the flow line).
9. Very slowly overlay each gel with 2.5 mL 95% ethanol. Allow it polymerization for 2 hours and then over with 2 ml of deionized water.
10. Cover the upper portion of the unit with wet paper towels and plastic wrap to prevent dehydration.
11. Allow polymerization for 16 hours.
12. Bring the unit near a sink, carefully disassemble, and scrape off access acrylamide into garbage bag. (Note:Start this step two hours before the completion of IPG focusing experiment).
13. Remove the gel cassette from the caster by pulling forward on the separator sheets, rinse the outer surface of each gel cassette with deionized water to remove any polyacrylamide particles, and place gel cassettes in the cassette rack.
14. Prepare stacking gel solution by combining 10.64 mL of acrylamide stock, 20 mL of 0.5 M Tris-HCl, pH 6.8, 0.8 mL of 10% SDS, and 48.8 mL of deionized water in a 200 mL beaker.
15. Mix thoroughly by stirring the solution on a stir plate.
16. Add 0.6 mL of 10% ammonium persulfate while stirring the solution.
17. Add 40 µL TEMED and continue to stir for 30 sec.
18. Place enough stacking gel solution on the top of the separating gel using a plastic transfer pipette to give 2 cm height after polymerization.
19. Overlay each gel with 1 mL isobutanol. (Note: Isobutanol is added to obtain uniform and straight layer on the gel which is necessary in order to place the IPG strip).
20. Allow to polymerize approximately one hour.
21. Once polymerized, pour off isobutanol and wash several times with deionized water to remove any traces of isobutanol.
22. Add 1 X SDS running buffer on the top of the stacking gel to prevent dehydration. (Note: It is important to keep the ready gels moist to prevent any dehydration. Gels can also be stored for a week at 4-8 °C, if wrapped with plastic wrap and moist paper towels to prevent dehydration ).
Reduction/Alkylation of Proteins in the IPG Strips
1. When the electrophoresis run has been completed, remove the IPG strips from the focusing tray by holding one end of the strip with forceps. Hold the IPG strips vertically with forceps and let the mineral oil drain to Kimwipes from the IPG strip for ~5 sec before transfer. .
3. Incubate on rocking platform at medium speed for 15 min at RT.
4. Transfer the IPG strips t o a new disposable tray with 2.5 mL alkylation solution in each well.
5. Incubate again on rocking platform at medium speed for 15 min at RT.
6. Transfer the IPG strips again to a new disposable tray carrying 2.5 mL 1 X SDS running buffer in each well for a brief wash. The IPG strips are now ready for SDS-PAGE.
1. Remove the IPG strip from the tray by holding one end of the strip with forceps and placing the IPG strip carefully on the surface of stacking gel. (Note: Add 1 X SDS running buffer on top of the stacking gels. It helps in sliding the strip between the plates and in positioning the strip on the gel surface. Again, avoid trapping air bubbles between strip and gel. The acidic (positive) end of the strip should be on the left. The gel face of the strip should not touch the opposite glass plate ).
2. Place a small square of electrode wick (0.5 cm ´ 0.5 cm) containing 8 µL of Wide range standards next to the acidic (positive) end of the strip. (Note:We recommend dipping the electrode wick containing PeppermintStick standards into the agarose sealing solution to prevent diffusion of standards into liquid. Usually we place electrode wick near the acidic end of the IPG strips. It is important to remove excess liquid before placing the electrode wick).
3. Quickly overlay the IPG strip and the electrode wick with 2-3 mL agarose overlay solution. (Note:Do not use too warm of agarose overlay solution. Again there should not be any air bubbles).
4. Pour about 7.5 L 1 X SDS running buffer into the lower chamber of the separation unit. (Note: The Ettan DALTtwelve electrophoresis unit requires a total of about 9.5 L of SDS running buffer. About 7.5 L 1 X SDS running buffer for the lower chamber and about 2.0 L of 2 X SDS running buffer for the upper chamber ).
5. Once agarose is solidified, insert the gel cassette into the separation unit through the buffer seal slots flanked by rubber gaskets. (Note:Unoccupied slots should be filled with the blank cassettes. Use 1 X SDS running buffer from a squirt bottle to wet the surface of gel or blank cassettes before inserting the cassettes, as it helps to slide easily into the uni t).
6. Pour about 2.0 L 2 X SDS running buffer until solution reaches the marked upper level on the separation unit.
7. Run the electrophoresis unit at 2 W per gel until dye migrates off the gel. (Note: It usually takes overnight [about 15-16 hours] to finish the run).