Hancock Lab

Immunoprecipitation

Categorized: Immunology Based Methods

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 37oC 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 4oC.
  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).

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