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:

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 (10,000 x g) 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 (saving supernatant).
Refrigerate, if the preparation is to be stored before use (e.g. SDS-PAGE).