title: Nonenzymatic chemiluminescent detection and quantitation of total protein on Western and slot blots allowing subsequent immunodetection and sequencing.
abstract: 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)-H2O2 reaction. Using this peroxyoxalate chemiluminescence system, the best results were obtained with proteins covalently labeled with 2-methoxy-2,4-diphenyl-3(2H)-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-H2O2 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.
authors.affiliation: Department de Bioquímica i Biologia Molecular, Facultat de Cièncias, Universitat Autònoma de Barcelona, Bellaterra, Spain.
authors.forename: F J
authors.initials: FJ
authors.lastname: Alba
authors.forename: J R
authors.initials: JR
authors.lastname: Daban
mesh_terms: D015153:Blotting, Western; D005456:Fluorescent Dyes; D005663:Furans; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000032:analysis; D007118:Immunoassay; D007768:Lactalbumin / Q000032:analysis; D007782:Lactoglobulins / Q000032:analysis; D008163:Luminescent Measurements; D010047:Ovalbumin / Q000032:analysis; D010070:Oxalates; D011506:Proteins / Q000032:analysis*; D017421:Sequence Analysis; D012710:Serum Albumin, Bovine / Q000032:analysis
chemical_list: D005456:Fluorescent Dyes; D005663:Furans; D007782:Lactoglobulins; D010070:Oxalates; D011506:Proteins; D012710:Serum Albumin, Bovine; C008394:2-methoxy-2,4-diphenyl-3(2H)-furanone; C044622:peroxyoxalate; D010047:Ovalbumin; D007768:Lactalbumin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases