Lissa1:Native Extraction: Difference between revisions
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# Add the appropriate amounts of pptase and/or pptase inhibitor to each aliquot: | # Add the appropriate amounts of pptase and/or pptase inhibitor to each aliquot: | ||
## 1.5 µL Pptase (Calf intestinal Phosphatase) | ## 1.5 µL Pptase (Calf intestinal Phosphatase) | ||
Revision as of 14:40, 3 July 2006
Pptase inhibitor protocol
- Freeze cell pellets in liquid nitrogen & Store –80°C
- Thaw cell pellets on ice
- Add 500 l ice-cold virgin lysis buffer and transfer to Bell lab notched screw-top tubes. Then add 500 l glass beads.
- Bead beat at 4°.
- 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, poke holes in bottom of tube with hot 21G needle. Spin.
- Make sure to tap tube to move beads to lid region
- Put holes in conical portion of tube
- Spin 5 min at 4° to pellet debris
- Recover lysate. Split into 50 µL aliquots, labeled N1 – N5, A1 – A5. Keep on ice!
- Add the appropriate amounts of pptase and/or pptase inhibitor to each aliquot:
- 1.5 µL Pptase (Calf intestinal Phosphatase)
- 22.5 µL pptase inhibitor (see below)
- Incubate 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
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
Running Gel (50 mL):
- 14.3 mL 3.5x gel buffer
- 20 mL 30% acrylamide /0.8% bis-acrylamide (12% running)
- 15.7 mL H2O
- Mix the above, then add
- 333 µL 10% APS (w/v)
- 93 µL TEMED
- Pour gel and cover with 1 mL of bis-Tris buffer saturated butanol
Stacking Gel (15 mL):
- 4.3 mL 3.5x gel buffer
- 2.5 mL 30% acrylamide /0.8% bis-acrylamide (5% stacking)
- 8.2 mL H2O
- 60 µL bromophenol blue
- Mix the above, then add:
- 75 (120) µL 10% APS (w/v)
- 37.5 (60) µL TEMED
Running buffer (2L)
- 104.5 g MOPS (MW = 209, 250 mM)
- 60.5 g Tris Base (MW = 121, 250 mM)
- 3.72 g EDTA (MW = 372.24, 5mM)
- 10 g SDS (0.5%)
- Add water to 2L
