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Role of ammonium persulphate in SDS-PAGE?
Ammonium persulphate is used in SDS-PAGE as a source of free radicals to initiate the polymerization of acrylamide and bisacrylamide monomers. When combined with a TEMED (Tetramethylethylenediamine) catalyst, it helps to create a crosslinked polyacrylamide gel matrix for separating proteins based on their size.
What's the function of TEMED?
Tetramethylethylenediamine is used with ammonium persulfate to catalyze the polymerization of acrylamide when making polyacrylamide gels, used in gel electrophoresis, for the separation of proteins or nucleic acids. Although the amounts used in this technique may vary from method to method, 0.1-0.2% v/v TMEDA is a "traditional" range.
Function of TEMED in sds page?
In SDS-PAGE, TEMED is used as an accelerator for the polymerization of acrylamide. It reacts with ammonium persulfate to generate free radicals, which initiate the crosslinking of acrylamide and bisacrylamide, resulting in the formation of a gel matrix. TEMED helps to ensure the proper formation of the gel for protein separation based on size.
What is the separating gel principle?
Good question! The basic idea is to cause particles of varying sizes to move through a gel made up of some physical 'mesh'. Common biological gels are made of Agarose proteins (for separating nucleic acids) or Acrylamide/bisAcrylamide 'mesh' for separating proteins. Smaller molecules can move through the network faster, and so will move farther in a particular time period (minute, half-hour, hour, whatever). So, if you run a gel for 30 minutes or so, you will have a distribution of sizes, with the smallest pieces out in front, and the larger ones progressively further behind. Often you will have these molecules moving in 'bands', which are clumps of lots of a molecule which are the same size. Usually, you get the proteins or nucleic acids to move by running a current across the gel, as these molecules have a slight electrical charge. If you also run a set of comparable molecules of known sizes (usually called a 'size standard') then you have something to compare your results to so you can tell what size a particular band is. You can have a 'separating gel' game yourself, if you want: set up an obstacle course (with tunnels and other barriers of various kinds), get about 30-50 people, with kids of all ages and adults of all ages, and have them all start at the same time. Then, when the first person gets about 2/3 of the way through, blow a whistle and have them all stop where they are. Then see how they're arranged - take a photo so you can have a visual record of your experiment!
What is the principle behind the SDS-PAGE?
SDS PAGE uses an anionic detergent (SDS) to denature proteins. the protein molecules become linearized. One SDS molecule binds to 2 amino acids. Due to this, the charge to mass ratio of all the denatured proteins in the mixture becomes constant. These protein molecules move in the gel (towards the anode) on the basis of their molecular weights only & are separated. The charge to mass ratio varies for each protein (in its native or partially denatured form). Estimation of molecular weight would then be complex. Hence, SDS denaturation is used. The gel matrix is formed of polyacrylamide. The polyacrylamide chains are crosslinked by N,N-methylene bisacrylamide comonomers. Polymerisation is initiated by ammonium persulfate (radical source) and catalysed by TEMED (a free radical donor and acceptor). The resolution & focus of the protein bands is increased by using discontinuous gels (Laemmli gels)- the stacking gel (pH 6.8, %T=3 to 5 %) & the resolving gel (pH 8.8, %T= 5 to 20 %). %T represents acrylamide percentage. These gels are usually run at constant current. At pH=6.8, most of the glycine in the population exist as zwitterions with no negative charge (pKa 1 =2.45; pKa 2 =9.6; pI=6.025). Only 0.0015% of the glycine is anionic at this pH (refer glycine titration curve & Henderson-Hasselbach equation). As such, bulk of the current is carried by the denatured, negatively charged, SDS-coated protein molecules. At this stage, the glycine ions lag behind the proteins. The order is as follows- chloride ions, denatured proteins, glycine ions. Upon entering the resolving gel (pH=8.8), the glycine zwitterions deprotonate to the anionic form. The proportion of these ions increase from 0.0015% to 15.8%. The carrying of the current is now shared by the ions such that protein molecules have a greater freedom to separate on the basis of molecular weights. Due to their small size, the glycine anions also tend to overtake the protein band, thus providing a sandwiching effect & greater resolution in the gel.
