A New Bromobimane Fluorescent Label for Anion Exchange Proteins
A new, negatively charged fluorescent labeling agent, sulfobenzoyloxybromobimane (SBBr), selectively labels the membrane anion exchange protein (Band 3) in intact erythrocytes under physiological conditions. The fluorescent product is stable and easily seen after electrophoretic separation of proteins from solubilized membranes (whole membranes or membranes stripped of peripheral proteins). Quantitation is carried out with solubilized membranes or by densitometry of the gel. Chloride ions diminish the rate of labeling. Diisothiocyanatostilbenedisulfonate (DIDS) inhibits labeling by SBBr. The major part of the label remains in the 60kDa fragment after chymotrypsin treatment of SBBr-labeled cells. SBBr-binding site(s) may be different from those attacked by DIDS. SBBr is useful for studying band 3 and related proteins, including C1- channels.
KeywordsCysteine Lysine Polypeptide Enzymatic Degradation Thiol
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