Protein Quantification
To quantify proteins, a standard curve was prepared by diluting a stock solution of BSA (Sigma) in double-distilled water at various concentrations varying from 10 to 50 mg/ml. Four volumes of each protein solution were mixed with one volume of dye reagent concentrate (Bio-Rad Laboratories) using a final volume of 1 ml of solution. The solutions were mixed and the optical density was monitored with a spectrophotometer (Molecular Devices) at a wavelength of 595 nm. A “blank” solution containing only the dye reagent diluted in double-distilled water as described above was used to subtract the optical density seen without proteins. A graph of optical density at 595 nm was plotted against the concentration of proteins from the standards. The samples with unknown concentration of proteins were processed the same way as the standard solutions. The protein concentration of the unknown was evaluated based on the optical density of the protein solutions and interpolation from the graph prepared. The solutions of unknown concentration were diluted until the optical density value obtained was within the linear part of the graph. The values of proteins mentioned represented average of experiments performed in triplicates.