Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis, commonly known as SDS-PAGE, is a very common technique used in the lab. It is used for the rough analysis of dirty (raw, impure) samples, sample separation and as a rough guide to protein quantity and identification. When used more carefully the technique can also be used to highlight protein weights, and thus roughly the lengths of the amino acid sequences that make up all proteins. But how does this experimental method work? Let’s find out!
SDS-PAGE is performed on a slippery, transparent and thin gel made up of cross-linked Acrylamide molecules and a number of other reagents; this produces a molecular mesh for the prepared sample to be teased through. The sample is first prepared by denaturing the proteins via boiling with Sodium Dodecyl Sulphate (SDS) Sample Buffer for about 10-15 minutes. The Sample Buffer contains a reagent that is ‘heavy’ (explained later on), colours the solution bright blue and can also contain mercaptoethanol, a chemical that breaks down di-sulphide bonds in multi-chain proteins to release the separate fragments. This boiling breaks down the complicated structure of the sample proteins to leave them in their primary structures (the simple amino acid sequence) and the SDS molecules can then attach to these chains and give a uniform, negative charge to the linearized proteins. This allows the prepared proteins to be attracted towards the positive cathode and pass through the pores of the gel at a rate relevant to their size when an electric current is passed through it. As has been mentioned, the gel is basically a molecular sieve so that larger proteins will blunder and struggle to move through the gel, thus migrating at a slower rate than faster, smaller proteins that can navigate through the pores better. This results in the separation of sample proteins by their size, and not on additional factors such as electric charge.
But an SDS-PAGE gel is not just a single flimsy bit of gel laid on a lab bench! Practically, the set-up of an SDS-PAGE gel is slightly more complicated. Think about it; by the time you’ve put in your last sample; your first sample will have diffused through the gel and already gotten a head-start on the others. To make sure all of the samples have an even start, a complete SDS-PAGE gel is actually made up of two gels- the Stacking gel for this purpose and Running gel- both made of the same reagents, just in different ratios. The Running gel is made first and is where the proteins will be teased and separated; its’ Acrylamide content varies depending on the proteins you are using, the more Acrylamide you add to the mixture the more cross-linking will happen and so the finer the sieve. This Running gel solution is pipetted in between two vertical, thin glass plates and allowed to solidify before the Stacking gel is made and pipetted ontop of the Running gel. A thin comb is stuck into the gap between the two plates before the Stacking gel solidifies; this is to create the sample wells that you will be putting your samples into, which will sink to the bottom of the well thanks to the ‘heavy’ Sample Buffer added previously. Once all of this has been allowed to settle, the comb is carefully taken out and the completed, sandwiched gel is put vertically into a casket and filled with Running Buffer which aids the flow of the electric current and keeps the gel moist. The lid of the casket is then put into place, and the whole set-up is plugged in, and allowed to run (Running times depend again on the proteins in question).
After the gel has been ran, the casket is opened, the glass-sandwiched gel taken out and the two plates carefully pried apart. The gel then undergoes a series of washes, staining and de-staining procedures (Coomasie Blue is commonly used) to stain the proteins in the gel more clearly and then to de-stain the rest of the gel for a nice visible contrast. After that, you can do what you like with the results!
And there you have it! A quick guide to the how’s and why’s of the SDS-PAGE technique, I hope it comes in useful!
Picture taken from https://ww2.chemistry.gatech.edu/~williams/bCourse_Information/4581/techniques/gel_elect/page_protein.html on 5/1/14 at 6.30pm.
Stellwagen NC (2009). Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution. Electrophoresis, 30 Suppl 1 PMID: 19517510