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

For two-dimensional gel electrophoresis of protein mixture derived from homogenized tumor tissue, treat with buffer containing chaotropes, zwitterionic detergent, and reducing agent. Chaotropes and detergent denature and solubilize the proteins. Reducing agents reduce the proteins' disulfide bonds, generating free thiol groups and unfolding the proteins for optimal protein resolution. 

Transfer the treated protein mixture into an immobilized pH gradient, IPG, strip holder. Place an IPG strip comprising linear pH gradient within the acrylamide gel matrix. Add mineral oil to prevent protein solution evaporation. Transfer the holder into an isoelectric focusing, IEF system. Allow the protein solution to rehydrate the strip.

Perform first-dimension IEF. The electric current causes charged proteins to migrate through the pH gradient towards oppositely-charged electrodes, immobilizing at their respective isoelectric points - pH at which the proteins have zero net charge.

Post-IEF, treat the IPG strip with sodium dodecyl sulfate, SDS, an anionic detergent that imparts negative charge to proteins. Equilibrate with an alkylating agent to modify the protein thiol groups, ensuring protein solubility during electrophoresis.

Load the strip onto a precast small-pore polyacrylamide-resolving gel and secure with molten agarose. Fill the chambers with SDS-containing electrophoresis buffer. Initiate second-dimension electrophoresis, perpendicular to IEF.

Negatively charged, larger molecular weight proteins move through the gel slowly towards the positively charged anode than smaller proteins, facilitating size-based separation.

Stain the gel. Proteins can be visualized as discrete spots on the gel.

To begin this procedure, add 250 microliters of a previously prepared protein sample solution to each slot of a 13-centimeter IPG strip holder. Place an 18-centimeter IPG strip, gel-side down, onto each protein sample solution, taking care to avoid bubbles. Then, add 3 to 4 milliliters of mineral oil to cover each IPG strip.

Next, assemble each IPG strip holder into an isoelectric focusing system, with the pointed end on the back plate, and the square end on the front plate. After rehydrating the IPG strips for 18 hours at room temperature, set the IEF parameters on the isoelectric focusing system with a total runtime of 7.5 hours and 36,875 volt hours.

After IEF, take out each IPG strip from the isoelectric focusing system, and remove the excess mineral oil with an insoluble paper towel. Then, wrap each strip in a sheet of plastic wrap, and store at -80 degrees Celsius.

To cast a 12% PAGE gel, add double-distilled water, 1.5 molar Tris-hydrochloric buffer, 40% acrylamide/bisacrylamide stock solution, 10% ammonium persulfate, and TEMED to a beaker. Mix the gel solution gently, taking care to avoid bubbles.

Gently pour the gel solution into a multi-casting chamber, up to the expected gel height of 19 centimeters. Immediately, add about 3 milliliters of double-distilled water to the multi-casting chamber to cover each resolving gel, and allow each gel to polymerize for about one hour.

This step is very important to prepare a high-quality SDS-PAGE gel. An equal volume water must be immediately added on the top of each gel.

Prepare two liters of electrophoresis buffer, then, add the buffer to an electrophoresis separation unit buffer tank.

After removing the focused IPG strips from the freezer, place them in a plastic vessel containing 1 milliliter of reducing equilibrium buffer and a trace of bromophenol blue, and equilibrate them for 10 minutes with gentle rocking. After removing the reducing equilibrium buffer, add 4 milliliters of alkylation equilibrium buffer, and a trace of bromophenol blue to the IPG strips, and equilibrate them for 10 minutes with gentle rocking.

During gel equilibrium, disassemble the multi-casting chamber, and remove the prepared resolving gel cassette. Rinse the gel cassette three times with double-distilled water, then, remove excess water from the gel cassette with an insoluble paper towel, and place it in a gel stander.

Following this, rinse the equilibrated IPG strips with electrophoresis buffer, and remove excess liquid on each IPG strip surface with an insoluble paper towel. Place an IPG strip onto the resolving SDS-PAGE gel, allowing the IPG strip's plastic side to contact the longer glass plate, with the pointed end to the left.

Now, quickly add 3 to 4 milliliters of hot 1% agarose and SDS electrophoresis buffer, to seal the IPG strip on the top of each SDS-PAGE gel, and place the top side of the IPG strip down on the top of the shorter glass plate, taking care to avoid bubbles.

These steps are very important to seal the IPG strip to an SDS-PAGE gel. The hot agarose solutions must be immediately added, and the IPG strip must be immediately placed into the top of the SDS gel.

After polymerization, insert the assembled gel cassette vertically between the plastic gaskets and a vertical electrophoresis system. Place the top of the gel with the IPG strip next to the cathode. Next, adjust the level of electrophoresis buffer to immerse the gel cassette by adding the appropriate volume of buffer to the electrophoresis system.

Connect and set the power supply in constant voltage mode, and run at 200 volts for about 370 minutes while monitoring the dye. After the run is complete, take the gel cassette out of the electrophoresis system and gently remove the gel from the gel cassette, taking care to avoid tearing the gel.