Pptase inhibitor protocol
- Freeze cell pellets in liquid nitrogen & Store –80°C
- Thaw cell pellets on ice
- Add 500 ul ice-cold virgin lysis buffer and transfer to Bell lab notched screw-top tubes. Then add 500 ul glass beads.
- Bead beat at 4 degrees by doing the following:
- Use Fast prep machine in the Bell Lab
- Balance machine with even number of tubes.
- Rotate starfish guard so it holds tube in place. Tighten with key.
- Speed: 6.5; Time: 45
- 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.
- Invert tubes, and tap tubes to beads move to lid region.
- Poke one hole in the bottom of each tube with hot 21G needle.
- To do this, heat the needle in a bunsen burner between pokes.
- Place tubes, hole side down, in plastic tubes (from Bell lab). Keep on ice, and move quickly to the next step.
- Spin 5 min at 3000 rpm at 4° to pellet debris. Use the swinging bucket centrifuge in the centrifuge room.
- Recover lysate (the liquid in the tube). Discard the screw-top tube. KEEP ON ICE!
- Split lysate into 50 µL aliquots in Epindorf tubes. Keep on ice!
- 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.
- 1.5 µL Pptase (Calf intestinal Phosphatase)
- 22.5 µL pptase inhibitor (see below)
- Incubate all samples at 37 °C for one hour.
- Add 50 µL of 2x sample buffer.
- Boil for 10 minutes.
- 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
- 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