Coomassie Brilliant Blue (CBB) is used in the Bradford assay to determine protein concentration. CBB forms a cationic, neutral, or anionic species depending on pH. In the assay, CBB exists primarily as a neutral species as the solution is acidic. Though the assay is widely used, it is not clear about CBB’s protein binding mechanism, especially the binding affinity (Ka) and Stoichiometry (n). Here, we used bovine serum albumin (BSA), typically used as a control for creating a standard curve, to study its interaction with CBB in solution with pH= ~1.5. Using UV/Vis spectrometry, a solution of BSA was titrated to CBB and the absorption increase at 615 nm was recorded. The data analysis by using alternated Scatchard’s plot yields the Ka and n values to be ~ 8.4 x105 M-1 and 1-2, respectively. To investigate the binding effect from the protein’s charged states, Calmodulin (CaM), consisting of several negatively charged residues, was used and its binding to CBB was studied. Our preliminary data showed that the binding is a sigmoidal curve, which indicated a cooperative binding mechanism. Currently, we are investigating how ammonium sulfate increases assay sensitivity.

Binding mechanism study of coomassie brilliant blue species and bovine serum albumin

253rd National Meeting (2017)

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