Lissa1:Native Extraction

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Pptase inhibitor protocol

  1. Freeze cell pellets in liquid nitrogen & Store –80°C
  2. Thaw cell pellets on ice
  3. Add 500 ul ice-cold virgin lysis buffer and transfer to Bell lab notched screw-top tubes. Then add 500 ul glass beads.
  4. Bead beat at 4 degrees by doing the following:
    1. Use Fast prep machine in the Bell Lab
    2. Balance machine with even number of tubes.
    3. Rotate starfish guard so it holds tube in place. Tighten with key.
    4. Speed: 6.5; Time: 45
  5. Once beating is finished, put tubes on ice. It is VERY important to keep tubes on ice from here on out, as the proteases will digest your proteins.
  6. Invert tubes, and tap tubes to beads move to lid region.
  7. Poke one hole in the bottom of each tube with hot 21G needle.
    1. To do this, heat the needle in a bunsen burner between pokes.
  8. Place tubes, hole side down, in plastic tubes (from Bell lab). Keep on ice, and move quickly to the next step.
  9. Spin 5 min at 3000 rpm at 4° to pellet debris. Use the swinging bucket centrifuge in the centrifuge room.
  10. Recover lysate (the liquid in the tube). Discard the screw-top tube. KEEP ON ICE!
  11. Split lysate into 50 µL aliquots in Epindorf tubes. Keep on ice!
  12. Add the appropriate amounts of pptase and/or pptase inhibitor to each aliquot. See sheet that Samantha sent you for samples to run. See below for the appropriate amounts of pptase or pptase inhibitor to add.
    1. 1.5 µL Pptase (Calf intestinal Phosphatase)
    2. 22.5 µL pptase inhibitor (see below)
  13. Incubate all samples at 37 °C for one hour.
  14. Add 50 µL of 2x sample buffer.
  15. Boil for 10 minutes.
  16. Load on gel.

Virgin Lysis Buffer (10 mL):

  • 1 mL HEPES buffer (pH = 7.5)
  • 10 µL 1 M MgCl2
  • 0.20% NP-40
  • 0.10% Triton X-100
  • 1.5 mL Cocktail stock solution
  • 7.47 mL H2O

CIP

  • 3 µL per sample (30 units)

Phosphatase inhibitor cocktail (310 µL ) (add 310 µL to 690 µL reaction buffer)

  • 100 µL 500 mM -glycerophosphate
  • 10 µL 200 mM sodium orthovanadate
  • 100 µL NaF
  • 100 µL Sodium pyrophosphate