Question

The following question is for Cellular and Molecular Biology lab.

1. What is bradford assay. Which dye do we use in Bradford assay? What wavelength we use to get readings for Bradford assay.

Answer 1

Bradford assay was discovered by Marion M. Bradford in 1976 . It is a procedure to measure the concentration of protein in a solution .

Principle for Bradford assay is the binding of the Coomassie Blue G250 dye to proteins . At pH 0 , both the sulfate are negatively charged and all three nitrogens are positively charged giving to dye +1 [ the red form of the dye ] . Around pH 1.5 , the neutral green form of the dye predominates . At neutral pH , the dye has a net charge of +1 [ the blue form of the dye ] . the red , green and bllue forms of the dye absorbs visible radiation with absorption maxima of 470 , 650 , 550 nm , respectively . It is the anionic form of the dye that binds to the protein . Binding of the blue form of Coomassie Blue G250 with protein causes red shift in its absorption spectrum ; the absorption shift maximum shift from 590 to 620 mm . Its clear to record the absorption at 620 mm . But the absorption is recorded at 595 mm to avoid any contribution of the green form . The dye binds more readily to the cationic residue , lysine and arginine . this clearly indicates that the response of the assay will depend on amino acid composition of protein , major drawback of this assay . Modification has been made to overcome this drawback .

COOMASSIE BLUE G250 dye we use in Bradford assay .

We use 595 nm wavelength to get readings for Bradford assay .