Isolation of hen oviduct ovalbumin and rat liver albumin polysomes by indirect immunoprecipitation

D. J. Shapiro, J. M. Taylor, G. S. McKnight, R. Palacios, C. Gonzalez, M. L. Kiely, R. T. Schimke

Research output: Contribution to journalArticle

Abstract

The polyribosomes synthesizing ovalbumin and albumin were 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 was formed by incubating antibody with polysomes, and then precipitated by reaction with an 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: incubation of oviduct polysomes containing labeled nascent peptide chains with antibovine serum albumin results in precipitation of only 0.4% of the labeled polysomes; indirect immunoprecipitates from oviduct polysomes reacted with antiovalbumin contain essentially no nascent peptide chains larger than ovalbumin; 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; 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.

Original languageEnglish (US)
Pages (from-to)3665-3671
Number of pages7
JournalJournal of Biological Chemistry
Volume249
Issue number12
StatePublished - Dec 1 1974
Externally publishedYes

Fingerprint

Polyribosomes
Oviducts
Ovalbumin
Immunoprecipitation
Liver
Rats
Albumins
Messenger RNA
Antibodies
Proteins
Peptides
Serum Albumin
Purification
Contamination
RNA

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Shapiro, D. J., Taylor, J. M., McKnight, G. S., Palacios, R., Gonzalez, C., Kiely, M. L., & Schimke, R. T. (1974). Isolation of hen oviduct ovalbumin and rat liver albumin polysomes by indirect immunoprecipitation. Journal of Biological Chemistry, 249(12), 3665-3671.

Isolation of hen oviduct ovalbumin and rat liver albumin polysomes by indirect immunoprecipitation. / Shapiro, D. J.; Taylor, J. M.; McKnight, G. S.; Palacios, R.; Gonzalez, C.; Kiely, M. L.; Schimke, R. T.

In: Journal of Biological Chemistry, Vol. 249, No. 12, 01.12.1974, p. 3665-3671.

Research output: Contribution to journalArticle

Shapiro, DJ, Taylor, JM, McKnight, GS, Palacios, R, Gonzalez, C, Kiely, ML & Schimke, RT 1974, 'Isolation of hen oviduct ovalbumin and rat liver albumin polysomes by indirect immunoprecipitation', Journal of Biological Chemistry, vol. 249, no. 12, pp. 3665-3671.
Shapiro DJ, Taylor JM, McKnight GS, Palacios R, Gonzalez C, Kiely ML et al. Isolation of hen oviduct ovalbumin and rat liver albumin polysomes by indirect immunoprecipitation. Journal of Biological Chemistry. 1974 Dec 1;249(12):3665-3671.
Shapiro, D. J. ; Taylor, J. M. ; McKnight, G. S. ; Palacios, R. ; Gonzalez, C. ; Kiely, M. L. ; Schimke, R. T. / Isolation of hen oviduct ovalbumin and rat liver albumin polysomes by indirect immunoprecipitation. In: Journal of Biological Chemistry. 1974 ; Vol. 249, No. 12. pp. 3665-3671.
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abstract = "The polyribosomes synthesizing ovalbumin and albumin were 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 was formed by incubating antibody with polysomes, and then precipitated by reaction with an 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: incubation of oviduct polysomes containing labeled nascent peptide chains with antibovine serum albumin results in precipitation of only 0.4{\%} of the labeled polysomes; indirect immunoprecipitates from oviduct polysomes reacted with antiovalbumin contain essentially no nascent peptide chains larger than ovalbumin; 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; 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.",
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