Immunoprecipitation of Proteins from Cell-Free Translations

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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