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Nonenzymatic chemiluminescent detection and quantitation of total protein on Western and slot blots allowing subsequent immunodetection and sequencing
Author(s) -
Alba F. Javier,
Daban JoanRamon
Publication year - 1997
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150181114
Subject(s) - chemiluminescence , chemistry , blot , peroxyoxalate , fluorescence , chromatography , oxalate , membrane , electrophoresis , biochemistry , organic chemistry , quantum mechanics , physics , gene
We have studied the light emission efficiency of proteins labeled with different fluorescent dyes chemically excited by the bis(2,4,6‐trichlorophenyl)oxalate (TCPO)‐H 2 O 2 reaction. Using this peroxyoxalate chemiluminescence system, the best results were obtained with proteins covalently labeled with 2‐methoxy‐2,4‐diphenyl‐3(2 H )‐furanone (MDPF). Blotted proteins on polyvinylidene difluoride (PVDF) membranes can be labeled rapidly with MDPF. Our results demonstrate that energy from the excited intermediate produced in the TCPO‐H 2 O 2 reaction can be efficiently transferred to MDPF‐labeled proteins in solution and on PVDF membranes. Although this nonenzymatic chemiluminescent system produces a background emission that reduces the sensitivity, the method developed in this work allows detection of 5 ng of protein in blots after 5 min exposure to X‐ray film. Chemiluminescence of MDPF‐labeled proteins on Western and slot blots may also be detected and quantified using a charge‐coupled device (CCD) camera or a storage phosphor imaging system. This chemiluminescent method allows the staining of the total electrophoretic pattern but does not preclude further N ‐terminal sequencing and immunodetection of specific bands.