This article outlines a protocol for protein separation using SDS-PAGE. The method involves denaturing proteins, loading them into a gel, and visualizing the results after electrophoresis.
Begin by treating the protein sample with a denaturing buffer comprising a detergent - sodium dodecyl sulfate, SDS, and a reducing agent - beta-mercaptoethanol. Heat it briefly. During this step, beta-mercaptoethanol disrupts the disulfide linkages, rendering an unfolded conformation to the protein.
The unfolded proteins with their exposed hydrophobic amino acids bind to the negatively charged SDS and acquire a uniform negative charge. Add a suitable tracking dye to the sample for real-time visualization.
Now, load the sample and standard molecular size marker in separate wells present in the stacking gel - the top layer of the polyacrylamide gel containing a lower concentration of cross-linked acrylamide polymers.
Connect the buffer-filled gel apparatus to the power supply.
In the presence of an electric field, negatively charged proteins migrate freely towards the anode and stack together to enter the resolving gel - the lower layer containing a higher concentration gel.
In the resolving gel, proteins start migrating as per their molecular size, with smaller proteins migrating rapidly, resulting in optimally resolved protein bands. Thereafter, stop the run and remove the gel.
Stain the gel with an anionic dye, Coomassie blue, that binds electrostatically to the basic amino acids in proteins, imparting them blue coloration. Thereafter, destain the gel in acetic acid solution for better visualization.
Remove the gel and identify the separated proteins based on their estimated molecular weights.
Prepare two separating gels as described in the text manuscript. Pour the gel between the glass plates using a 1-milliliter pipette, ensuring that the upper 2 centimeters are free of the mixture. Add 70% ethanol on top of the separating gel, creating an even interface between the two layers.
Once the separating gels have polymerized, prepare the stacking gels using instructions in the text manuscript. Then, remove the ethanol from the separating gels, and add the stacking gel solution. Carefully insert a comb with the desired number of pockets, without introducing air bubbles, and allow the gel to polymerize for 20 to 30 minutes.
Load 4 microliters of each sample, as well as the protein ladder, into separate wells. Then, run the gel in Tris-Glycine running buffer at 144 volts for 45 minutes at room temperature. Use pre-warmed Fairbanks solution A to stain the gels on a rocker for 30 minutes, and pre-warmed Fairbanks solution D to decolor the gels on a rocker until the desired background is achieved.
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