What is the principle behind the SDS-PAGE?

Answer 1

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.