[8] Immunoprecipitation of proteins from cell-free translations

doi:10.1016/S0076-6879(83)96012-3

Methods in Enzymology

Volume 96, 1983, Pages 111-120

https://doi.org/10.1016/S0076-6879(83)96012-3 Get rights and content

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.

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[8] Immunoprecipitation of proteins from cell-free translations

Abstract

J. Biol. Chem.

J. Mol. Biol.

Cell

Cell

J. Immunol. Methods

J. Biol. Chem.