Simultaneous immunoblotting analysis with activity gel electrophoresis in a single polyacrylamide gel
Resource
ELECTROPHORESIS 22 (10): 1894-1899
Journal
Electrophoresis
Journal Volume
22
Journal Issue
10
Pages
1894-1899
Date Issued
2001
Date
2001
Author(s)
Chen, Hungwen
Abstract
We describe here that a simple diffusion blotting method can couple immunoblotting analysis with another biochemical technique in a single polyacrylamide gel. The efficiency of protein transfer was evaluated by serial dilutions of nephrosin, a metalloproteinase of the astacin family, and by immunodetection. It is estimated that diffusion blotting produces 25-50% of the signal intensity compared to the classical electrophoretic transfer method. However, with diffusion blotting it is possible to generate several replicas from a single gel. In addition, a protein blot can be obtained from a sodium dodecyl sulfate (SDS)-polyacrylamide gel for zymography assay or from a native polyacrylamide gel for electrophoretic mobility shift assay (EMSA). In this regard, a particular signal in zymography or EMSA can be confirmed by simultaneous immunoblotting analysis with a corresponding antiserum. Therefore, diffusion blotting allows a direct comparison of signals between gels and replicas in zymography assay and EMSA. These advantages make diffusion blotting desirable when partial loss of transfer efficiency can be tolerated or be compensated by a more sensitive immunodetection reaction using enhanced chemiluminescence substrates.
Subjects
Electrophoretic mobility shift assay; Multiple blotting; Proteinase; Western blotting; Zymography
Other Subjects
antiserum; dodecyl sulfate sodium; metalloproteinase; polyacrylamide gel; protein; analytic method; article; chemoluminescence; controlled study; diffusion; dilution; electrophoretic mobility; gel mobility shift assay; immunoblotting; polyacrylamide gel electrophoresis; protein determination; protein transport; zymography; Animals; Autoradiography; Blotting, Western; Carps; Electrophoresis, Polyacrylamide Gel; Humans; Metalloendopeptidases; NF-kappa B; Proteins
Type
journal article
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