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 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.
Steinberg
T H
TH
Molecular Probes, Eugene, OR 97402, USA.
Lauber
W M
WM
Berggren
K
K
Kemper
C
C
Yue
S
S
Patton
W F
WF
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D011817:Rabbits; D012965:Sodium Chloride / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005456:Fluorescent Dyes; D012996:Solutions; D012967:Sodium Dodecyl Sulfate; D012965:Sodium Chloride