Immunoprecipitation of proteins from cell-free translations.
Immunoprecipitation of proteins from cell-free translations.
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DOI：
10.1016/s0076-6879(83)96012-3
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发表时间：
1983
影响因子：
--
通讯作者：
D. Anderson;G. Blobel
中科院分区：
文献类型：
--
作者：
D. Anderson;G. Blobel
在小麦胚芽无细胞系统中合成的蛋白质（丰度约0.5%）的典型免疫沉淀程序总结如下。1.将2微升的25%SDS加入到48微升的翻译反应混合物中，并将样品加热至100度持续4分钟。向上述样品中添加四体积（即200微升）4度稀释缓冲液。稀释缓冲液为1.25% Triton X-100、190 mM NaCl、60 mM Tris-HCl（pH 7.4）、6 mM EDTA、10单位特雷西洛/毫升。3.加入5微升适当的抗血清，并将样品在4 ℃下孵育至少12小时。4.将样品在微量离心机中旋转2分钟，并将上清液转移至新管中。5.加入30微升蛋白质A-琼脂糖CL-4 B的1：1悬浮液（15微升填充珠），并将样品在室温下端对端混合孵育2小时。通过在微量离心机中离心10秒使琼脂糖凝胶珠粒沉淀，并吸出上清液。7.将珠粒在室温下在每毫升1 ml 0.1%Triton X-100、0.02%SDS、150 mM NaCl、50 mM Tris-HCl（pH 7.5）、5 mM EDTA、10单位Trasylol中洗涤四次，每次洗涤时涡旋。8.用上述不含洗涤剂的溶液对珠粒进行最后洗涤，并用抽出的巴斯德移液管尽可能完全地吸出上清液。9.将40微升含有50 mM DTT的SDS-凝胶电泳样品缓冲液加入珠中，并将样品在沸水浴中加热4分钟。10.通过在样品缓冲液中加入10微升1.0M碘乙酰胺并在37 ℃下孵育45分钟来封闭游离-SH基团。11.离心出珠粒，并将上清液施加到SDS-聚丙烯酰胺平板凝胶上。
A typical procedure for immunoprecipitating a protein (abundance ca 0.5%) synthesized in the wheat germ cell-free system is summarized below. 1. Two microliters of 25% SDS are added to 48 microliters of translation reaction mixture, and the sample is heated to 100 degrees for 4 min. 2. Four volumes (ie, 200 microliters) of dilution buffer at 4 degrees 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. Five microliters of appropriate antisera are added, and the sample is incubated for at least 12 hr at 4 degrees. 4. The sample is spun for 2 min in a microcentrifuge, and the supernatant is transferred to a fresh tube. 5. Thirty microliters of a 1: 1 suspension of protein A-Sepharose CL-4B (15 microliters of packed beads) are added, and the sample is incubated with end-over-end mixing at room temperature for 2 hr. 6. The Sepharose beads are pelleted by a 10-sec centrifugation in the microcentrifuge, and the supernatant is aspirated. 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. 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. 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. Free--SH groups are blocked by adding 10 microliters of 1.0 M iodoacetamide in sample buffer and incubating for 45 min at 37 degrees. 11. The beads are centrifuged out, and the supernatant is applied to an SDS-polyacrylamide slab gel.