- The persulfate ion in ammonium persulfate is the equivalent of two sulfate ions linked by a weak oxygen-oxygen bond. This bond can cleave or break in two, yielding sulfate radicals --- highly reactive molecules with an unpaired electron. The sulfate radical may react with water to generate a hydroxyl radical, or it may react with the acrylamide monomer to start the polymerization reaction. TEMED acts as a catalyst that accelerates the rate at which persulfate ions form radicals.
- Once a hydroxyl or sulfate radical has formed, it attacks an acrylamide monomer, taking one of the electrons in the double bond. The acrylamide monomer is now a radical with an unpaired electron, so it goes on to attack another acrylamide monomer in a similar fashion. This series of events creates a fast-growing chain of acrylamide monomers linked by single bonds. The chain reaction only ends when two radicals react with each other.
- If acrylamide were present on its own, it wouldn't form the thick gel you need for the SDS-PAGE. The polymers have to be cross-linked to form a gel with the right properties, and that's where bisacrylamide comes in. The double bonds in bisacrylamide also serve as a target for radicals in the reaction, and because it has two sites where it can be attacked by radicals, it's ultimately incorporated into two of the growing polymers. Consequently, the polymers end up with crosslinks between them at random sites along the length of each chain.
- The polymerization reaction is very rapid, so once you've added the TEMED and the persulfate, it will take place very quickly. The purity of your reagents is important because contaminants can affect your results. Acrylic acid, for example, can be added to the polymer and creates ion-exchange sites that might cause local pH variations in your gel. The resulting gel may still contain some acrylamide monomer, and acrylamide is a neurotoxin, so always take appropriate precautions when working with SDS gels.
Initiation
Chain Reaction
Cross-Linking
Considerations
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