A modified PABC immunoassay for the quantitation of DNA dependent RNA polymerase I: a procedure applicable to other proteins present in minute amounts and/or isoforms.
An indirect enzyme linked immunoassay (ELISA) has been developed to measure the amount of RNA polymerase I (E.C.2.7.7.6) in silkmoth tissue cell extracts. Subunit specific monoclonal antibodies (MABs) were immobilized on the solid substrate by a variation of the widely used Protein-Avidin-Biotin-Capture (PABC) technique. The use of the commercially available biotinylated anti-mouse antibody as a bridge to bind the monoclonal antibody eliminates the need for the biotinylation of the monoclonal antibody in the laboratory. The RNA polymerase in solution was captured by the monoclonal antibody and was measured by the successive binding of rabbit polyclonal antibody and alkaline phosphatase conjugated anti-rabbit antibody. This procedure is more reliable, reproducible and leads to greater sensitivity compared to the direct binding of the monoclonal antibody to the microtiter plate. RNA polymerase I captured by the antibodies from tissue extracts was measured at levels of 0.5 ng/well. This assay system can be utilized as a general procedure to quantitate the levels of proteins present at very low levels and that are found in different isoforms containing multiple and/or shared subunits.
Mattes
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Department of Biochemistry and Molecular Biology, Texas Tech University Health Sciences Center, Lubbock 79430.
Sridhara
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001360:Avidin; D001426:Bacterial Proteins; D001710:Biotin; D012831:Bombyx / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005088:Exocrine Glands / Q000201:enzymology; D007074:Immunoglobulin G / Q000276:immunology; D051379:Mice; D012318:RNA Polymerase I / Q000032:analysis* / Q000276:immunology; D012319:RNA Polymerase II / Q000276:immunology; D012320:RNA Polymerase III / Q000276:immunology; D015203:Reproducibility of Results; D019809:Streptavidin; D014158:Transcription, Genetic; D014921:Wings, Animal / Q000201:enzymology
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D007074:Immunoglobulin G; D001360:Avidin; D001710:Biotin; D019809:Streptavidin; D012319:RNA Polymerase II; D012318:RNA Polymerase I; D012320:RNA Polymerase III