Two-layer antibody capture of enzymes on the surface of microtiter plates: application to the study of the regulation of phospholipase C-γ1 catalytic activity
| Autor: | Colin T Buckley, Kevin K. Caldwell |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Gene isoform
Time Factors Biophysics Phospholipase Biochemistry Antibodies Chemistry Techniques Analytical Rats Sprague-Dawley Animals Molecular Biology chemistry.chemical_classification Chromatography biology Phospholipase C Phospholipase C gamma Cell Biology Enzyme assay In vitro Enzymes Rats Enzyme chemistry Type C Phospholipases Biotinylation biology.protein Female Tyrosine kinase |
| Zdroj: | Analytical Biochemistry. 320:193-198 |
| ISSN: | 0003-2697 |
| Popis: | In vitro quantification of the catalytic activity of an enzyme isoform requires the availability of selective agents that allow for either measurements in the presence of the other enzyme isoforms or purification of the isoform and subsequent performance of these measurements on the purified enzyme. Isozyme-specific antibodies are useful tools for these types of analyses. In the present report, we detail a method for the measurement of phospholipase C-gamma1 enzyme activity employing native enzyme that is immobilized on microtiter plates. The method uses biotinylated antiglobulin bound to streptavidin-coated microtiter plates to immobilize antiphospholipase C-gamma1 antibody and subsequently capture phospholipase C-gamma1 from brain tissue lysates. This method avoids biotinylation of the primary (antiphospholipase C-gamma1) antibody, making it less labor intensive than previously described methods for using streptavidin-coated plates; in addition, it is highly reproducible and sensitive and allows for quantification of enzyme activity. We employ the technique to show that one or more tyrosine kinases copurify with rat brain phospholipase C-gamma1. The method is applicable to the study of any enzyme isoform for which antibodies that capture the native form of the enzyme are available and could easily be employed in high-throughput procedures. |
| Databáze: | OpenAIRE |
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