Immunoprecipitation
互联网
Hancock Laboratory Methods.Department of Microbiology and Immunology,
University of British Columbia,British Columbia,Canada
http://www.cmdr.ubc.ca/bobh/showmethod.php?methodid=28
MATERIALS:
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 |
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 37℃ for 10 to 15 minutes.
Pellet cell debris in an Eppendorf mini centrifuge (10,000 x g)for 3 minutes.
Decant the supernatant into a fresh tube and add 40-50 μl of antiserum.
Incubate on ice for 2 hours or overnight at 4℃.
Add 100 μl of S.aureus protein A and 100 μl 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 μl running buffer.
Boil in a water bath for 90 seconds and centrifuge for 1 minute to remove S.aureus (saving supernatant).
Refrigerate,if the preparation is to be stored before use (e.g.SDS-PAGE).
