Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution

SYPRO Tangerine stain is an environmentally benign alternative to conventio nal protein stains that does not require solvents such as methanol or aceti c acid for effective protein visualization. Instead, proteins can be staine d in a wide range of buffers, including phosphate-buffered saline or simply 150 mM NaCl using an easy, one-step procedure that does not require destai ning. Stained proteins can be excited by ultraviolet light of about 300 nm or with visible light of about 490 nm. The fluorescence emission maximum of the dye is approximately 640 nm. Noncovalent binding of SYPRO Tangerine dy e is mediated by sodium dodecyl sulfate (SDS) and to a lesser extent by hyd rophobic amino acid residues in proteins. This is in stark contrast to acid ic silver nitrate staining, which interacts predominantly with lysine resid ues or Coomassie Blue R, which in turn interacts primarily with arginine an d lysine residues. The sensitivity of SYPRO Tangerine stain is similar to t hat of the SYPRO Red and SYPRO Orange stains - about 4-10 ng per protein ba nd. This detection sensitivity is comparable to colloidal Coomassie blue st aining and rapid silver staining procedures. Since proteins stained with SY PRO Tangerine dye are not fixed, they can easily be eluted from gels or uti lized in zymographic assays, provided that SDS does not inactivate the prot ein of interest. This is demonstrated with in-gel detection of rabbit liver esterase activity using a-naphthyl acetate and Fast Blue BB dye as well as Escherichia coli beta-glucuronidase activity using ELF-97 beta-D-glucuroni de. The dye is also suitable for staining proteins in gels prior to their t ransfer to membranes by electroblotting. Gentle staining conditions are exp ected to improve protein recovery after electroelution and to reduce the po tential for artifactual protein modifications such as the alkylation of lys ine and esterification of glutamate residues, which complicate interpretati on of peptide fragment profiles generated by mass spectrometry.