SDS-PAGE

In SDS (sodium dodecyl sulfate)-PAGE, the proteins are coated and denatured with a whole negative charge [due to bound sodium dodecyl sulfate (SDS) molecules] and thus the basis for their separation is only their mass. The protein combination  is  first  treated  with  a  reducing  agent  like  as  dithiothreitol  or2-mercaptoethanol to break all the disul?de bonds. The tough anionic detergent sodium dodecyl sulfate is then added that disrupts nearly all the noncovalent interactions in the protein, unfolding the polypeptide chain. Around  one molecule  of  sodium dodecyl sulfate binds  by  its  hydrophobic   alkyl  chain  to  the  polypeptide

Figure: Polyacrylamide gel electrophoresis. The protein samples are loaded into the sample wells formed in the top of the gel. An electric field is applied across the gel from top to bottom and the proteins migrate down through the gel. The smaller the protein the further it will migrate.

Figure: SDS-PAGE. The protein mixture is heated in the presence of 2-mercaptoethanol, which breaks any disul?de bonds, and SDS. The unfolded polypeptide chains are coated with the  negatively-charged molecules  of  SDS  and  will  migrate  towards  the  anode  on polyacrylamide gel electrophoresis. Smaller polypeptides migrate further through the gel than larger ones.

backbone for each two amino acid residues, that gives the denatured protein a large  net negative  charge  which  is proportional  to its mass.  The SDS or protein combination is then applied to sample wells in the top of a polyacrylamide gel in the figure. The buffer, that is the similar in both the lower and upper reservoirs  and in the gel, has a pH of around  9, like in which the proteins  have net negative charge  and will migrate  towards  the anode  in the lower  reservoir.  An electric current (around 300 V) is applied across the gel from top to bottom for 30–90 min in order to move the proteins by the gel in figure. After carrying out electrophoresis the gel is erased from the proteins and the apparatus described. Small proteins move furthest by the gel, while large ones move more slowly as they are held back through the cross-linking in the gel. Under these situations, the mobility of most polypeptide chains is linearly proportional to the logarithm of their mass. Therefore, if proteins  of known  molecular mass are electrophoresed  alongside  the templates the mass of the unknown proteins can be determined as there is a linear relationship among log10  of molecular  distance  and mass migrated  by  the gel. Proteins which differ in mass through about 2 percent (for instance 40 and 41 kDa; a difference of around 10 amino acid residues) can be illustrious under appropriate conditions.  SDS- PAGE  is a sensitive, rapid and  broadly-used  method  that  can  be  used  to determine  the degree  of purity  of a protein  sample  to estimate  the molecular mass of an unknown protein and to deduce the number of polypeptide subunits within a protein.