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

Abstract

SYPRO Tangerine stain is an environmentally benign alternative to conventional protein stains that does not require solvents such as methanol or acetic acid for effective protein visualization. Instead, proteins can be stained 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 destaining. 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 dye is mediated by sodium dodecyl sulfate (SDS) and to a lesser extent by hydrophobic amino acid residues in proteins. This is in stark contrast to acidic silver nitrate staining, which interacts predominantly with lysine residues or Coomassie Blue R, which in turn interacts primarily with arginine and lysine residues. The sensitivity of SYPRO Tangerine stain is similar to that of the SYPRO Red and SYPRO Orange stains - about 4-10 ng per protein band. This detection sensitivity is comparable to colloidal Coomassie blue staining and rapid silver staining procedures. Since proteins stained with SYPRO Tangerine dye are not fixed, they can easily be eluted from gels or utilized in zymographic assays, provided that SDS does not inactivate the protein of interest. This is demonstrated with in-gel detection of rabbit liver esterase activity using alpha-naphthyl acetate and Fast Blue BB dye as well as Escherichia coli beta-glucuronidase activity using ELF-97 beta-D-glucuronide. The dye is also suitable for staining proteins in gels prior to their transfer to membranes by electroblotting. Gentle staining conditions are expected to improve protein recovery after electroelution and to reduce the potential for artifactual protein modifications such as the alkylation of lysine and esterification of glutamate residues, which complicate interpretation of peptide fragment profiles generated by mass spectrometry.

An improved formulation of SYPRO Ruby protein gel stain: Comparison with the original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation

“…Exposure times were generally 1 to 8 s. RuBPSA was imaged in an identical manner as SYPRO Ruby protein gel stain on the indicated instruments. Fluorescence detection of glycoproteins and b-glucuronidase activity was performed as described previously [26,35,36]. Briefly, either 490 nm long pass or 520 nm band pass emission filters are suitable for imaging these dyes.…”

“…Staining for b-glucuronidase activity in gels was accomplished by previously described procedures [26,36]. Briefly, after electrophoresis, gels were incubated in two 10 min.…”

Fluorescent labeling of proteins with Nile red and 2-methoxy-2,4-diphenyl-3(2H)-furanone: Physicochemical basis and application to the rapid staining of sodium dodecyl sulfate polyacrylamide gels and Western blots

“…This hydrophobic environment could be responsible of the remarkable fluorescence enhancement that allows the visualization of protein bands in SDS-polyacrylamide gels (see below). This mechanism could also operate with other hydrophobic dyes that yield intense fluorescence intensities with protein-SDS complexes [10,11]. The amino acid composition may modulate the structure of protein-SDS complexes and/or the affinity of different dyes for these complexes.…”

“…Fluorescent staining of polyacrylamide gels depends on the interaction of Nile red with protein-SDS complexes [9]. New fluorescent dyes, SYPRO orange, red and tangerine [10,11], capable of interacting with these complexes, have been developed. These and other noncovalent fluorescent dyes useful for the staining of polyacrylamide-SDS gels have been recently reviewed [12,13].…”

Detection of biomolecules in electrophoresis gels with salts of imidazole and zinc II: A decade of research

“…A variety of staining methods [2][3][4][5] are available for the nonradioactive localization of the separated bands. They may be based on (i) selective binding of organic dyes (e.g., Coomassie blue dye) to biomolecules, (ii) noncovalent binding of fluorescent groups (e.g., Sypro dyes, ethidium bromide) followed by gel irradiation with UV light, (iii) reduction of silver salts to metallic silver, or (iv) selective precipitation of metal cations on the gel matrix (negative staining) or in the zones occupied by biomolecules (positive staining).…”