SDS-PAGE

SDS-PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis) is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 kDa. The combined use of sodium dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and polyacrylamide gel allows to eliminate the influence of structure and charge, and proteins are separated solely on the basis of differences in their molecular weight. SDS-PAGE is an electrophoresis method that allows protein separation by mass. The medium (also referred to as ′matrix′) is a polyacrylamide-based discontinuous gel. The polyacrylamide-gel is typically sandwiched between two glass plates in a slab gel. Although tube gels (in glass cylinders) were used historically, they were rapidly made obsolete with the invention of the more convenient slab gels. In addition, SDS (sodium dodecyl sulfate) is used. About 1.4 grams of SDS bind to a gram of protein, corresponding to one SDS molecule per two amino acids. SDS acts as a surfactant, masking the proteins’ intrinsic charge and conferring them very similar charge-to-mass ratios. The intrinsic charges of the proteins are negligible in comparison to the SDS loading, and the positive charges are also greatly reduced in the basic pH range of a separating gel. Upon application of a constant electric field, the protein migrate towards the anode, each with a different speed, depending on its mass. This simple procedure allows precise protein separation by mass. SDS tends to form spherical micelles in aqueous solutions above a certain concentration called the critical micellar concentration (CMC). Above the critical micellar concentration of 7 to 10 millimolar in solutions, the SDS simultaneously occurs as single molecules (monomer) and as micelles, below the CMC SDS occurs only as monomers in aqueous solutions. At the critical micellar concentration, a micelle consists of about 62 SDS molecules. However, only SDS monomers bind to proteins via hydrophobic interactions, whereas the SDS micelles are anionic on the outside and do not adsorb any protein. SDS is amphipathic in nature, which allows it to unfold both polar and nonpolar sections of protein structure.[8] In SDS concentrations above 0.1 millimolar, the unfolding of proteins begins, and above 1 mM, most proteins are denatured. Due to the strong denaturing effect of SDS and the subsequent dissociation of protein complexes, quaternary structures can generally not be determined with SDS. Exceptions are proteins that are stabilised by covalent cross-linking e.g. -S-S- linkages and the SDS-resistant protein complexes, which are stable even in the presence of SDS (the latter, however, only at room temperature). To denature the SDS-resistant complexes a high activation energy is required, which is achieved by heating. SDS resistance is based on a metastability of the protein fold. Although the native, fully folded, SDS-resistant protein does not have sufficient stability in the presence of SDS, the chemical equilibrium of denaturation at room temperature occurs slowly. Stable protein complexes are characterised not only by SDS resistance but also by stability against proteases and an increased biological half-life. Alternatively, polyacrylamide gel electrophoresis can also be performed with the cationic surfactants CTAB in a CTAB-PAGE, or 16-BAC in a BAC-PAGE. Problem: The molecular weight of an unspecified protein, at physiological conditions, is 70,000 Dalton, as determined by sedimentation equilibrium measurements and by gel filtration chromatography. The SDS-polyacrylamide gel electrophoresis (SDS PAGE) of the protein yields a single band corresponding to molecular weight of 70,000 Dalton. However, in the presence of the reducing agent, β-mercaptoethanol, the SDS PAGE shows two bands, corresponding to molecular weights of 30,000 and 20,000 Dalton. From these data, describe the native protein in terms of the number of subunits present, their molecular weight, stoichiometry of subunits, and the kinds of bonding (covalent, noncovalent) existing between the subunits. #GelElectrophoresis #SDSPAGE #protein #polypeptide #DNA #mRNA #Dalton #molecularWeight