Two-Dimensional Polyacrylamide Gel Electrophoresis: A Separation Technique for Analysis of Proteins in Complex Protein Mixture Based on Charge and Molecular Weight

Protocol

1. Preparation of Samples

1. Collect pituitary adenoma tissues (0.2 - 0.5 mg) from the neurosurgical department. Immediately freeze in liquid nitrogen, and then transfer to -80 °C for storage.
2. Add 2 mL of 0.9% NaCl in deionized distilled water (ddH₂O). Use this solution to lightly wash the blood from the tissue surface (3x).
   NOTE: ddH₂O with a conductivity of 18.2 MΩ/cm is used throughout the protocol. Some of the tissue might be lost when washing off blood from a tissue's surface.
3. Add a volume (10 mL; 4 °C) of the solution containing 2 M acetic acid and 0.1% (v/v) β-mercaptoethanol for every 0.5 - 0.6 g of tissue, homogenize (1 min, 13,000 rpm, 4 °C, 10x) with a tissue homogenizer, sonicate the homogenate for 20 s, lyophilize, and store at −80 °C.
4. Measure the protein content of the lyophilized, homogenized samples using bicinchoninic acid (BCA) assay kit.
   NOTE: BCA quantification is not an absolute quantification, and the measured result will be altered with a different technician and the experimental agent. A fixed concentration sample standard should be used for different experiments. Therefore, the final loading amount of proteins for each 2D gel will be determined with the silver-stained or Coomassie-stained good-resolution image in the pre-designed experiments.
   1. Add about 300 μg of the lyophilized homogenized sample to one volume (282 μL) of protein-extracting buffer (8 M urea and 4% CHAPS), followed by standing for 2 h, sonicating in a water bath for 5 min, rotating for 1 h, sonicating again in the water bath for 5 min, rotating again for 1 h, and centrifuging at 15,000 x g for 20 min.
   2. Prepare BSA standard solutions with concentrations as mentioned: 0, 25, 125, 250, 500, 750, 1,500, 2,000 μg/mL with the commercial BSA standard (2 mg/mL).
   3. Mix BCA reagent A and B (A:B = 50:1) as BCA working solution prior to use.
   4. Add 0.1 mL of the sample or the standard solution to 2 mL of BCA working solution in a microfuge tube, followed by mixing and then incubating at 37 °C for 30 min. Finally cool at room temperature for 10 min and measure A_{562 nm} O.D. value.
   5. Calculate the standard linear line (A_{562 nm} vs. BSA concentration) to obtain a regression equation for calculating the sample protein content with A_{562 nm} value.
5. Use 150 μg of the protein equivalent lyophilized sample for silver staining, or 500 μg of protein for Coomassie staining, for an 18-cm immobilized pH gradient (IPG) strip pH 3-10 nonlinear (NL).
   NOTE: The IPG dry strip is 0.5-mm thick and 3-mm wide, with different lengths including 7, 11, 13, 18, and 24 cm, and different pH ranges including pH 4-5, 4-7, 6-9, and 3-10 in either a linear or nonlinear pH gradient. The IPG buffer used must fit the strip.
6. Add 250 μL of extracting buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 100 mM DTT (add prior to use), 0.5% v/v pharmalyte (add prior to use), and a trace of bromophenol blue).
7. Keep the solution below 30 °C, followed by vortexing for 5 min, sonicating for 5 min, and rotating for 50 min.
8. Add 110 μL of rehydration buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 60 mM DTT (add prior to use), 0.5% v/v IPG buffer (add prior to use), and a trace of bromophenol blue). Sonicate for 5 min. Then rotate the sample for 50 min, vortex for 5 min, and centrifuge for 20 min at 15,000 x g.
9. Collect the supernatant (350 μL) as the protein sample solution.

2. Two-Dimensional Gel Electrophoresis

1. IEF (First dimension): Perform IEF on the isoelectric focusing system as described below.
   1. Add 350 μL of the protein sample solution in the slot of an 18-cm IPG strip holder.
   2. Put an 18-cm IPG strip gel-side-down onto the protein sample solution (avoid bubbles).
   3. Add 3-4 mL of mineral oil to cover the IPG strip.
   4. Assemble the IPG strip holder into the isoelectric focusing system with the pointed end on the back (+) plate and square end on the front (−) plate.
   5. Rehydrate overnight (~18 h at room temperature).
   6. Set the IEF parameters: maximum current 30 μA per strip, 20 °C; 250 V, 1 h, 125 Vh, step and hold; 1,000 V, 1 h, 500 Vh, gradient; 8,000 V, 1 h, 4,000 Vh, gradient; 8,000 V, 4 h, 32,000 Vh, step and hold; and 500 V, 0.5 h, 250 Vh, step and hold. Let it run up to a total time of 7.5 h and 36,875 Vh.
   7. After IEF, take out each IPG strip and remove the extra mineral oil with an insoluble paper towel. Now wrap the strip in a sheet of plastic wrap, and store at −80 °C.
      NOTE: The IPG strip holder should be cleaned with the IPG strip holder cleaning solution and distilled water.
2. SDS-PAGE (Second dimension): Perform SDS-PAGE in a Vertical Cell Electrophoresis System
   NOTE: Each SDS-PAGE gel size should be 255 x 190 x 1 mm.
   1. Use a multi-gel caster to cast 12% PAGE resolving gels. For casting 12% PAGE gels, perform the following steps.
      1. Add 270 mL of ddH₂O, 150 mL of 1.5 M Tris-HCl (pH 8.8), 180 mL of 40% (w/v) acrylamide/bisacrylamide stock solution (29:1, 40% w/v acrylamide and 1.38% w/v N, N'-methylenebisacrylamide), 3 mL of 10% ammonium persulfate, and 150 μL of TEMED to make the gel solution in a beaker. Mix gently and avoid bubbles.
      2. Pour the gel solution gently into the multicasting chamber up to the expected gel height (19 cm).
      3. Immediately add about 3 mL ddH₂O on each resolving gel to cover the gels. Let the gel polymerize for about 1 h.
   2. Prepare 20 L of electrophoresis buffer (25 mM Tris, 192 mM glycerine, and 0.1% SDS). Fill the electrophoresis separation unit buffer tank with this buffer.
   3. Take out the focused IPG strips from the freezer and equilibrate the strips for 10 min by rocking gently in 4 mL of reducing equilibrium buffer (375 mM Tris-HCl (pH 8.8), 6 M urea, 2% (w/v) SDS, 20% (v/v) glycerol, 2% w/v DTT (add prior to use), and a trace of bromophenol blue).
   4. Equilibrate for 10 min by gently rocking the reduced IPG strips in 4 mL alkylation equilibrium buffer (375 mM Tris-HCl (pH 8.8), 6 M urea, 2% (w/v) SDS, 20% (v/v) glycerol, 2.5% w/v iodoacetamide (add prior to use), and a trace of bromophenol blue).
   5. During gel equilibrium, disassemble the multicasting chamber, and take out the prepared resolving gel cassette. Rinse 3x with ddH₂O. Remove excess ddH₂O with an insoluble paper towel and put in a gel stander.
   6. Rinse the equilibrated IPG strips with electrophoresis buffer and remove excess liquid on the IPG strip surface with the insoluble paper towel.
   7. Put an IPG strip onto the resolving SDS-PAGE gel and let the IPG strip's plastic side contact the longer glass plate and the pointed end to the left.
   8. Pour 3-4 mL of hot 1% agarose in SDS electrophoresis buffer (~80 °C) quickly to seal the IPG strip on the top of each SDS-PAGE gel, and put the top-side of IPG strip down to the top of the shorter glass plate without bubbles, and then polymerize for 10 min.
   9. Insert the assembled gel cassette vertically between plastic gaskets in the vertical electrophoresis system. Put the top of the gel with the IPG strip next to the cathode (−).
   10. Adjust the level of electrophoresis buffer to immerse the gel cassette.
   11. Connect and set the Power Supply/Control Unit in constant voltage mode and run at 200 V for about 370 min while monitoring the dye.
   12. After running, take out the gel cassette from the electrophoresis system, and gently remove the gel from the gel cassette, avoiding tearing the gel, followed by staining of 2DE-separated proteins.

Materials

Name	Company	Catalog Number	Comments
Ettan IPGphor 3	GE Healthcare		Isoelectric focusing system.
Ettan DALT II System	Amersham Pharmacia Biotech, Piscataway, NJ, USA		The vertical electrophoresis system
Ettan IPGphor strip holder	Amersham Pharmacia Biotech, Piscataway, NJ, USA
Ettan DALTsix multigel caster	Amersham Pharmacia Biotech, Piscataway, NJ, USA		Multigel caster
KimWipe	Kimvipe		Insoluble paper towel
Water	Made by PURELAB flex instrument
Polytron Model P710/35 homogenizer	Brinkmann Instruments, Westbury, NY
Pierce BCA Protein Assay Kit	Thermo Fisher Scientific	23227
2-D Quant Kit	GE Healthcare	80-6483-56
BIS-ACRYLAMIDE	AMRESCO	0172
ACRYLAMIDE	AMRESCO	0341
DTT	Sigma-Aldrich	D0632
Thiourea	Sigma-Aldrich	T8656
Urea	VETEC	V900119
SDS	AMRESCO	0227
CHAPS	AMRESCO	0465
TEMED	AMRESCO	M146
Ammonium Persulfate	AMRESCO	M133
IPG buffer pH 3-10, NL	GE Healthcare	17-6000-87
Immobiline Dry Strip pH 3-10NL, 18cm	GE Healthcare	17-1235-01