IMMUNOPRECIPITATION
MATERIALS:
Running buffer:
| 2ul |
1.25M Tris-HCl, pH 6.8 |
|
35ul |
distilled water |
|
2.5ul |
2-mercaptoethanol |
|
12.5ul |
10% SDS |
|
10ul |
80% glycerol |
|
2ul |
bromphenol blue |
Make up running buffer fresh before use.
TNE buffer:
| 10mM Tris-HCL, pH 8.0 |
|
10mM NaCl |
|
0.5mM EDTA |
METHODS:
- Resuspend cell pellet in TNE containing 1% NP40 and vortex.
- Incubate for 30 minutes on ice or at 37oC for 10 to 15 minutes.
- Pellet cell debris in an Eppendorf centrifuge for 3 minutes.
- Decant the supernatant into a fresh tube and add 40-50ul of antiserum.
- Incubate on ice for 2 hours or overnight at 4oC.
- Add 100ul of S. aureus protein A and 100ul 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 70ul 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.
