Covalent immobilization of proteins for high-sensitivity sequence analysis: electroblotting onto chemically activated glass from sodium dodecyl sulfate-polyacrylamide gels.
We report a new method for the preparation of proteins in a form suitable for high-sensitivity N-terminal amino acid sequence analysis. Proteins separated by polyacrylamide gel electrophoresis were electrophoretically transferred onto glass fiber filter paper chemically activated by the introduction of phenyl isothiocyanate functional groups. The proteins became covalently coupled to the matrix during the electrotransfer process. Bands containing transferred proteins were detected by fluorescent staining or autoradiography, cut out from the glass fiber filter, and directly loaded into the cartridge of a gas-phase sequenator. The covalent nature of the interactions between protein and glass fiber support permitted the use of more vigorous solid-phase sequencing protocols and of alternative sequencing reagents. This high-efficiency isolation and covalent coupling method provides the essential first step toward enhanced-sensitivity protein sequence analysis. The method has been successfully applied to the isolation of a wide variety of proteins from SDS-polyacrylamide gels, and was shown to be compatible with both the standard Edman reagent phenyl isothiocyanate and alternative sequencing reagents such as 4-(N,N'-dimethylamino)azobenzene-4'-isothiocyanate (DABITC).
Aebersold
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RH
Division of Biology, California Institute of Technology, Pasadena 91125.
Pipes
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Nika
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Hood
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Kent
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D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005898:Glass; D011506:Proteins / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate
D011506:Proteins; D012967:Sodium Dodecyl Sulfate