Bradford assay is a rapid and accurate method to determine the concentration of protein. The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. Within the linear range of the assay (~5-25 mg/ml), the more protein present, the more Coomassie binds.
Coomassie Blue R (CBR) is sensitive for protein detection in PAGE gels, and protein can be directly quantitated by colorimetric. Coomassie Blue dye binds to peptides in an acidic medium. The color change of this assay is measured at 595 or 600 nm. We calculate the protein concentration using the formula created by standards. This AbVideo displays the procedure of protein quantification by using CBR.
Steps of quantifying protein using UV absorbance (280nm) are demonstrated. 280nm absorbance depends on the Tyr and Trp (amino acids with aromatic rings) content. A very rough protein concentration can be obtained by making the assumption that the protein sample has an extinction coefficient of 1 (1 OD = 1 mg/ml protein).
Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It combines excellent sensitivity while using very simple and cheap equipment and chemicals. The procedures of silver staining are protein fixation, silver impregnation and image development.