Immunoprecipitation

Materials:

Running buffer:

• 2 µl 1.25M Tris-HCl, pH 6.
• 35 µl distilled water
• 2.5 µl 2-mercaptoethanol
• 12.5 µl 10% SDS
• 10 µl 80% glycerol
• 2 µl 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:

1. Resuspend cell pellet in buffer TNE containing 1% NP40 detergent and vortex.
2. Incubate for 30 minutes on ice or at 37^oC for 10 to 15 minutes.
3. Pellet cell debris in an Eppendorf mini centrifuge (10,000 x g) for 3 minutes.
4. Decant the supernatant into a fresh tube and add 40-50 µl of antiserum.
5. Incubate on ice for 2 hours or overnight at 4^oC.
6. Add 100 µl of S. aureus protein A and 100 µl 5% BSA in TNE.
7. Incubate on ice for 2 hours.
8. 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.
9. Resuspend the final pellet in 70 µl running buffer.
10. Boil in a water bath for 90 seconds and centrifuge for 1 minute to remove S. aureus (saving supernatant).
11. Refrigerate, if the preparation is to be stored before use (e.g. SDS-PAGE).