Question

Global regulators are proteins that can repress or activate the expression of large numbers of genes in an organism. A recent report describes structural studies on the anti-repressor AbbA from the bacterium Bacillus subtilis [Tucker et aL, J. Mol. Biol. (2014)]. This protein has the ability to bind to a second protein, a global repressor named AbrB.

These researchers first characterized their recombinant version of AbbA, a 65-residue protein prepared in E.coli cells.

(Qa).During the isolation and purification of AbbA, samples were monitored using a combination of SDS-PAGE gels, Bradford and BCA assays. Identify the general chemical properties utilized for protein measurements by 1. Bradford and 2. BCA assays.In each case, comment on the effectiveness of the particular assay for monitoring protein concentration.

(Qb).What specific information is obtained for a protein sample from an SDS-PAGE gel that cannot be obtained from the Bradford or BCA procedures?

Answer 1

Qa) Bradford assay is based on the binding of the protein to a dye known as coomassie blue g250 under acidic conditions. the formation od protein-dye complex results in formation of blue color, theintensity fo which can be quantified spectrophotometrically at 595nm. In the assay, the presence of basic amino acids such as lysine, arginine , histidine are measured. This assay is more useful where there is presence of reducing agents that do not interfere much. However, presence of detergents inthe protein sample can interfere with protein quantification.

BCA assay, also known as bicinnchoninic assay is also a protein estimation assay. This is based on the reduction of cupric ions into cuprous ions by the proteins containing cystine, tyrosine and tryptophan mainly in basic environment. the reduced copper ions then forma complex with two BCA molecules resulting a purple colored product which can be quantified spectrophotometrically at 562nm. This assay is very sensitve and can measure protein concentrations ranging from 5ug/ml to 2mg/ml. This assay is not preferred when the sample contains reducing agents.

Qb)Using an SDS PAGE , the molecular weight of the protein can be determined.