| Autor: |
Mary L. Kiely, Robert T. Schimke, Claudio F. Gonzalez, John M. Taylor, Rafael Palacios, G. S. McKnight, David J. Shapiro |
| Rok vydání: |
1974 |
| Předmět: |
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| Zdroj: |
Journal of Biological Chemistry. 249:3665-3671 |
| ISSN: |
0021-9258 |
| DOI: |
10.1016/s0021-9258(19)42526-x |
| Popis: |
The polyribosomes synthesizing ovalbumin and albumin have been isolated from total oviduct polysomes and total rat liver polysomes, respectively. The isolation was performed using an indirect immunoprecipitation technique which is based on the specific reaction which occurs between antibody against a purified native protein and the nascent peptide chains on polysomes synthesizing that protein. A soluble antibody-nascent chain-polysome complex is formed by incubating antibody with polysomes, and then precipitated by reaction with an anti-antibody. The techniques developed are both efficient and highly specific. Near quantitative isolation of polysomal mRNA coding for a specific protein may be achieved. Indirect immunoprecipitation results in nonspecific precipitation of less than 0.5% of total polysomes. Ovalbumin mRNA isolated by indirect immunoprecipitation is 99% pure with respect to contamination by other species of mRNA and rat liver albumin mRNA is greater than 95% pure. Evidence supporting these conclusions includes: (a) incubation of oviduct polysomes containing labeled nascent peptide chains with anti-bovine serum albumin results in precipitation of only 0.4% of the labeled polysomes; (b) indirect immunoprecipitates from oviduct polysomes reacted with anti-ovalbumin contain essentially no nascent peptide chains larger than ovalbumin; (c) ovalbumin and albumin mRNAs extracted from immunoprecipitates are enriched for the synthesis of their respective proteins by 1.8- and 8.7-fold, exactly the degree of purification predicted from their relative rates of synthesis; (d) isolation of albumin synthesizing polysomes from a mixture of rat liver polysomes and hen oviduct polysomes resulted in nonspecific precipitation of only 0.4% of the ovalbumin synthesizing polysomes when the immunoprecipitated RNA was assayed for its ability to synthesize ovalbumin in vitro. Indirect immunoprecipitation appears to be a general method widely applicable to the separation and isolation of polysomes and messenger RNAs coding for specific proteins. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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