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Immunoprecipitation of Proteins from Cell-Free Translations

Anderson, David J. and Blobel, Günter (1983) Immunoprecipitation of Proteins from Cell-Free Translations. In: Biomembranes, Part J: Membrane Biogenesis: Assemby and Targeting (General Methods: Eukaryotes), 96. Methods in Enzymology. No.96. Academic Press , New York, NY, pp. 111-120. ISBN 978-0-12-181996-5. https://resolver.caltech.edu/CaltechAUTHORS:20150415-084901208

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Abstract

A typical procedure for immunoprecipitating a protein (abundance ca 0.5%) synthesized in the wheat germ cell-free system is summarized below. 1. 1. Two microliters of 25% SDS are added to 48 μl of translation reaction mixture, and the sample is heated to 100° for 4 min. 2. 2. Four volumes (i.e., 200 μl) of dilution buffer at 4° are added to the above sample. Dilution buffer is 1.25% Triton X-100, 190 mM NaCl, 60 mM Tris-HCl, pH 7.4, 6 mM EDTA, 10 units of Trasylol per milliliter. 3. 3. Five microliters of appropriate antisera are added, and the sample is incubated for at least 12 hr at 4°. 4. 4. The sample is spun for 2 min in a microcentrifuge, and the supernatant is transferred to a fresh tube. 5. 5. Thirty microliters of a 1:1 suspension of protein A—Sepharose CL-4B (15 μl of packed beads) are added, and the sample is incubated with end-over-end mixing at room temperature for 2 hr. 6. 6. The Sepharose beads are pelleted by a 10-sec centrifugation in the microcentrifuge, and the supernatant is aspirated. 7. 7. The beads are washed four times in 1 ml, per wash, of 0.1% Triton X-100, 0.02% SDS, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 10 units of Trasylol per milliliter at room temperature with vortexing at each wash. 8. 8. The beads are given a final wash with the above solution not containing detergent, and the supernatant is aspirated as completely as possible with a drawn-out Pasteur pipette. 9. 9. Forty microliters of SDS-gel electrophoresis sample buffer containing 50 mM DTT are added to the beads, and the sample is heated for 4 min in a boiling water bath. 10. 10. Free—SH groups are blocked by adding 10 μl of 1.0 M iodoacetamide in sample buffer and incubating for 45 min at 37°. 11. 11. The beads are centrifuged out, and the supernatant is applied to an SDS-polyacrylamide slab gel.


Item Type:Book Section
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/S0076-6879(83)96012-3DOIArticle
http://www.sciencedirect.com/science/article/pii/S0076687983960123PublisherArticle
ORCID:
AuthorORCID
Anderson, David J.0000-0001-6175-3872
Additional Information:© 1983 by Academic Press, Inc.
Series Name:Methods in Enzymology
Issue or Number:96
Record Number:CaltechAUTHORS:20150415-084901208
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20150415-084901208
Official Citation:David J. Anderson, Günter Blobel, [8] Immunoprecipitation of proteins from cell-free translations, In: Sidney Fleischer, Becca Fleischer, Editor(s), Methods in Enzymology, Academic Press, 1983, Volume 96, Pages 111-120, ISSN 0076-6879, ISBN 9780121819965, http://dx.doi.org/10.1016/S0076-6879(83)96012-3. (http://www.sciencedirect.com/science/article/pii/S0076687983960123)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:56665
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:16 Apr 2015 04:18
Last Modified:09 Mar 2020 13:18

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