Nonenzymatic chemiluminescent detection and quantitation of total protein on Western and slot blots allowing subsequent immunodetection and sequencing.
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.
Alba
F J
FJ
Department de Bioquímica i Biologia Molecular, Facultat de Cièncias, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Daban
J R
JR
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
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