Question

Explain how the protein samples usually need to be processed before applying the sample to the polyacrylamide gel and the assay can be performed.

Why is a molecular weight standard always applied in the same polyacrylamide gel as the protein samples?

How can the analysis show us that the protein sample is pure, ie consists of only one type of protein?

Answer 1

1. Protein samples usually need to be processed before applying the sample to the polyacrylamide gel and the assay can be performed . The steps for the smaple prepration are as followed:-

protein samples are denatured by heating it for 3 to 5 minutes in the presence of chemical denaturent 1% of SDS (sodium do- decyl sulphate) with or without reducing agent such as 20mM DTT, 2-mercaptoethanol (BME) or Tris. This SDS is an anionic detergent that denatures the secondary and non disulfide linked tertiary structures, and applies a negative charge to each protein in proportion to its mass. Then , it is cooled at room temperature. These reducing agent further denatures the proteins by reducing disulfide linkages and thus overcoming some forms of tertiary protein folding, and breaking up quaternary protein structure.

Then a suitable negatively charged, low-molecular weight dye like bromophenol blue is added in the sample buffer which have higher electrophoretic mobility than the analytes so it migrates at buffer front and allow the experimenter to track the progress of the solution through the gel during the electrophoretic run.

2. Molecular weight standard is always applied in the same polyacrylamide gel as the protein samples because the ionic detergent SDS denatures and binds to proteins therby making them uniformly negatively charged. so, when a current is applied, all SDS-bound proteins in a sample will migrate through the gel toward the positively charged electrode depending upon their molecular mass. The proteins with lower mass will move more quickly than those with greater mass therby producing the band on the gel.

3.  If the result would show a single protein band after SDS-PAGE separation it means that the protein sample is pure and it consists of only one type of protein.If this would not have been the case and there would have been the multiple protein in the samples, then multiple protein bands should have appeared through polyacrylamide gel.