In vivo cross-linking

  1. Inoculate 3 ml minimal media with single colonies from appropriate strains. Incubate 8 hours at 30oC.

  2. Inoculate 33 ml minimal media from previous cultures. Incubate at 30oC until O.D.(600) = 1 (~2E7 cells/ml).

  3. Harvest cells by 5 minute centrifugation in table top centrifuge.

  4. Remove excess medium by decanting, not aspiration.

  5. Add 3.3 ml XL buffer + 80 ul 5 mg/ml zymolase. Resuspend cells.

  6. Divide into 3 - 1 ml aliquots in 2 ml eppendorf tubes.

  7. Add 20 ul of appropriate concentration of DSP (prepared fresh in DMSO) to each aliquot. Vortex briefly.

  8. Incubate 40 minutes at 30oC on a nutator making sure the cell suspension moves freely within the tube.

  9. Pellet spheroplasts by microcentrifugation at 14,000 rpm for 3 minutes.

  10. Remove supernatant by decanting, not aspiration.

  11. Add 300 ul XSUMED + protease inhibitors.

  12. Add 100 ul scoop of Acid Washed Glass Beads.

  13. Lyse on multivortexer for 3 minutes (speed setting 7 on our current machine).

  14. Using a 1ml blue pipet tip, remove the cell lysate to a new tube.

  15. Clarify the lysate by centrifugation at 21,000 x g for 15 minutes.

  16. Remove supernatant to conical 1.5 ml eppendorf tube.

    1. Add antiserum (Ab) to stock of IP buffer (1ml IP buffer per sample) + protease inhibitors.

    2. Add 1 ml antiserum (Ab)/IP mixture to supernatant from cross-linking aliquots.

    3. Nutate 12 to 15 hours at 4oC or 2 to 3 hours at room temp.

    4. Add 100 ul (10% w/v) ProteinA Sepharose to the mix. DON'T VORTEX!!!

    5. Incubate another 2 hours at room temperature.

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

    7. 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. Aspirate beads to dryness with 25G (blue) fine gauge needle inserted into the beads.

    8. Add 50 ul SUMEB buffer to ProteinA-Sepharose beads and incubate at 65oC, or other desired temperature, for 10 minutes.

    9. Load on 7 ul or 30 ul on appropriate percentage SDS-PAGE gel.

    10. 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.


    XL BUFFER
    (1.2 M Sorbitol, 5 mM EDTA, 0.1 M KH2PO4/K2HPO4 pH 7.5))

    TO MAKE 500 ml:

    • 109.3 g sorbitol
    • 5.0 ml 0.5 M EDTA stock solution
    • 10.0 ml 1 M KH2PO4
  17. 40.0 ml 1 M K2HPO4
    Should be pH 7.5. I usually make a 1M KH2PO4/K2HPO4 buffer first by mizing the two until I get the desired pH. Then add 50 ml of that to the above stock.

    XSUMED BUFFER
    (1% SDS, 1% Triton X100, 0.5% Deoxycholate, 8 M Urea, 10 mM MOPS, pH 6.8, 10 mM EDTA)

    TO MAKE 100 ml:

    • 1.0 ml 1M 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
    • 1.0 ml Triton X100
  18. 0.5 g Deoxycholate

    SUMEB BUFFER
    (1% SDS, 8 M Urea, 10 mM MOPS, pH 6.8, 10 mM EDTA, 0.01% bromophenol blue)

    TO MAKE 100 ml:

    • 1.0 ml 1M 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
    • 1.0 ml 1% bromophenol blue


    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:

      • 0.213 g Na2HPO4
      • 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.