Immunoprecipitation

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:

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).