$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
1. Preparation of Samples
- 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.
- 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.
- 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.
- 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.
- 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.
- 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).
- Mix BCA reagent A and B (A:B = 50:1) as BCA working solution prior to use.
- 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 A562 nm O.D. value.
- Calculate the standard linear line (A562 nm vs. BSA concentration) to obtain a regression equation for calculating the sample protein content with A562 nm value.
- 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.
- 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).
- Keep the solution below 30 °C, followed by vortexing for 5 min, sonicating for 5 min, and rotating for 50 min.
- 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.
- Collect the supernatant (350 μL) as the protein sample solution.
2. Two-Dimensional Gel Electrophoresis
- IEF (First dimension): Perform IEF on the isoelectric focusing system as described below.
- Add 350 μL of the protein sample solution in the slot of an 18-cm IPG strip holder.
- Put an 18-cm IPG strip gel-side-down onto the protein sample solution (avoid bubbles).
- Add 3-4 mL of mineral oil to cover the IPG strip.
- 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.
- Rehydrate overnight (~18 h at room temperature).
- 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.
- 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.
- 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.
- Use a multi-gel caster to cast 12% PAGE resolving gels. For casting 12% PAGE gels, perform the following steps.
- 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.
- Pour the gel solution gently into the multicasting chamber up to the expected gel height (19 cm).
- Immediately add about 3 mL ddH2O on each resolving gel to cover the gels. Let the gel polymerize for about 1 h.
- 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.
- 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).
- 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).
- 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.
- Rinse the equilibrated IPG strips with electrophoresis buffer and remove excess liquid on the IPG strip surface with the insoluble paper towel.
- 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.
- 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.
- 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 (−).
- Adjust the level of electrophoresis buffer to immerse the gel cassette.
- 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.
- 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.