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Immunoprecipitation

来源:http://www.17930ip.com 作者:威尼斯城所有登入网址 时间:2020-02-07 20:04

核心提示:MATERIALS: Running buffer:2 ul1.25M Tris-HCl, pH 6.35 uldistilled water2.5 ul2-mercaptoethanoMATERIALS:

Running buffer:

2 ul 1.25M Tris-HCl, pH 6.
35 ul distilled water
2.5 ul 2-mercaptoethanol
12.5 ul 10% SDS
10 ul 80% glycerol
2 ul bromphenol blue

Make up running buffer fresh before use.

TNE buffer:

10 mM Tris-HCL, pH 8.0 10 mM NaCl 0.5 mM EDTA

METHODS:

Resuspend cell pellet in buffer TNE containing 1% NP40 detergent and vortex. Incubate for 30 minutes on ice or at 37oC for 10 to 15 minutes. Pellet cell debris in an Eppendorf mini centrifuge for 3 minutes. Decant the supernatant into a fresh tube and add 40-50 ?l of antiserum. Incubate on ice for 2 hours or overnight at 4oC. Add 100 ?l of S. aureus protein A and 100 ?l 5% BSA in TNE. Incubate on ice for 2 hours. Pellet the immune complexes and wash twice with 1ml 1%NP40, 0.5% Na deoxychloate, 0.1% SDS in TNE and sonicate once to resuspend. Resuspend the final pellet in 70 ?l running buffer. Boil in a water bath for 90 seconds and centrifuge for 1 minute to remove S. aureus . Refrigerate, if the preparation is to be stored before use .

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