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IMMUNOPRECIPITATION

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:

  1. Resuspend cell pellet in TNE containing 1% NP40 and vortex.
  2. Incubate for 30 minutes on ice or at 37oC for 10 to 15 minutes.
  3. Pellet cell debris in an Eppendorf centrifuge for 3 minutes.
  4. Decant the supernatant into a fresh tube and add 40-50ul of antiserum.
  5. Incubate on ice for 2 hours or overnight at 4oC.
  6. Add 100ul of S. aureus protein A and 100ul 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 70ul 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.