Antibody-linked polymerase assay on protein blots: a novel method for identifying polymerases following SDS-polyacrylamide gel electrophoresis.
We describe a method for correlating polymerase activity with a particular polypeptide band in an SDS-polyacrylamide gel which does not require renaturation of the SDS-denatured enzyme. The method involves the following steps: (i) transfer of proteins from an SDS-polyacrylamide gel onto nitrocellulose; (ii) incubation with excess antiserum raised against a partially purified polymerase preparation to link one Fab site of an antibody molecule to the denatured enzyme on the nitrocellulose; (iii) binding of native polymerase to the other Fab site of the antibody molecule in the immune complex to generate a specific polymerase 'sandwich'; (iv) assaying of the nitrocellulose filter for antibody-linked native polymerase activity using an appropriate template and a radioactive substrate followed by treatment with trichloroacetic acid to precipitate in situ the radioactive product. The essential feature of this method is that the use of both non-specific anti-polymerase serum and a partially purified enzyme preparation is sufficient to allow identification of a specific protein following SDS-polyacrylamide gel electrophoresis. This antibody-linked polymerase assay has been developed to identify a 130,000-dalton RNA-dependent RNA polymerase from cowpea leaves. Possible applications of this type of assay as a tool for identifying a wide variety of proteins are discussed.
van der Meer
J
J
Department of Molecular Biology, Agricultural University, Wageningen, The Netherlands.
Dorssers
L
L
Zabel
P
P
D000818:Animals; D000906:Antibodies / Q000276:immunology; D017798:Comovirus / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D015151:Immunoblotting / Q000379:methods; D012324:RNA-Dependent RNA Polymerase / Q000276:immunology / Q000378:metabolism*; D011817:Rabbits; D012967:Sodium Dodecyl Sulfate; D013379:Substrate Specificity; D014544:Uridine Triphosphate
D000906:Antibodies; D012967:Sodium Dodecyl Sulfate; D012324:RNA-Dependent RNA Polymerase; D014544:Uridine Triphosphate