Protein Immunoprecipitation from a Cell Lysate

  1. Grow desired volume of cultures to 1 O.D.(600) (~2E7 cells/ml) using selective media.

  2. Harvest cells by centrigugation.

  3. Lyse cells as follows:

    1. Harvest 2.0 ml cells in a round bottom eppendorf by spinning full speed for 3 minutes in a microcentrifuge.

    2. Decant and discard supernatant.

    3. Add 200 ul of SUME buffer + protease inhibitors.

    4. Add 1 100 ul scoop of 0.5 mm Acid Washed Glass Beads.

    5. Vortex in multivortexer for 3 minutes (speed setting 7 on our current machine).

    6. Remove the lysate from the beads with a blue pipette tip to a new 1.5 ml conical bottom eppendorf tube.

    7. Spin 5 minutes to clarify. Remove supernatant to a new 1.5 ml conical bottom eppendorf tube.

  1. Add 1.0 ml IP buffer to each sample.

  2. Add 10 ul antibody to each sample, and incubate at 4oC overnight.

  3. Add 100 ul of 10% ProteinA-Sepharose to each sample, and incubate at room temperature for 2 hours.

  4. Spin samples at 1000 rpm in microcentrifuge for 15 seconds, then carefully remove lysate and save in a new 1.5 ml eppendorf tube.

  5. Wash ProteinA-Sepharose beads once with IP buffer and twice with wash buffer by resuspending beads in buffer, incubating 1 minute and microcentrifuging at 1000 rpm for 15 seconds. Apsirate the supernatant from the beads.

  6. Add 50 ul SUME buffer + 0.005% bromophenol blue to ProteinA-Sepharose beads and incubate at 65oC, or other desired temperature, for 10 minutes.

  7. Load 10-30 ul samples onto a SDS-PAGE gel.

  8. After running gel, transfer proteins to nitrocellulose and perform a western.

Note that protease inhibitors are added from stocks to give 50 fold dilution.
Example: 20 ul of stock per 1ml buffer.

Use IMMEDIATELY after adding protease inhibitors.


SUME BUFFER
(1% SDS, 8 M Urea, 10 mM MOPS, pH 6.8, 10 mM EDTA)

TO MAKE 100 ml:

  • 1.0 ml 1 M MOPS, pH 6.8 stock solution
  • 1.0 g SDS, or 10 ml 10% SDS stock solution
  • 48.05 g Urea
  • 2.0 ml 0.5 M EDTA stock solution This is used for lysing yeast at pH 6.8. For pH 8 lyses, use the variation of SUME with Tris buffer known as SUTE.

    Immunoprecipitation (IP) Buffer
    (15 mM Na2HPO4, mw 142; 150 mM NaCl, mw 58; 2% Triton X-100, 0.1% SDS, 0.5% DOC, 10 mM EDTA, 0.02% NaN3)

    TO MAKE 100 ml:

    • 0.213 g Na2HPO4
    • 0.87 g NaCl
    • 2.0 ml TritonX-100
    • 0.1 g SDS or 1 ml 10% SDS stock solution
    • 0.5 g deoxycholate
    • 2.0 ml 0.5M EDTA stock solution
    • 0.2 ml 10% NaN3 stock solution (optional) Final pH should be 7.5. Sterilize through a sterile filter if azide (NaN3) is not added.
      Wear a mask while handling the SDS. Requires Heat to get SDS into solution.

      Wash Buffer
      (50 mM NaCl, mw58; 10 mM TRIS, mw 121; 0.02% NaN3)

      TO MAKE 100 ml:

      • 0.29 g NaCl
      • 0.12 g Tris
      • 0.2 ml 10% NaN3 stock solution (optional)

      Final pH should be 7.5. Sterilize through a sterile filter if azide (NaN3) is not added.

      50X STOCK Protease Inhibitors (store at -20oC)

      PMSF (87 mg/ml)
      ([500 mM] phenylmethylsulfonyl fluoride)

      TO MAKE 30 ml:  Dissolve 2.61 g PMSF in DMSO to equal a final volume of 30 ml.

      LEUPEPTIN AND PEPSTATIN (5 mg/ml)

      TO MAKE 10 ml:  Dissolve 50 mg LEUPEPTIN or PEPSTATIN in DMSO to equal a final volume of 10 ml.

      TPCK (5 mg/ml)
      (tosylphenylalanine chloromethyl ketone)

      TO MAKE 10 ml:  Dissolve 50 mg TPCK in DMSO to equal a final volume of 10 ml.


      * Note: The EDTA in the SUME buffer is also a protease inhibitor.