Lissa1:Native Extraction

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:
 * 5) Use Fast prep machine in the Bell Lab
 * 6) 	Balance machine with even number of tubes.
 * 7) 	Rotate starfish guard so it holds tube in place. Tighten with key.
 * 8) 	Speed: 6.5; Time: 45
 * 9) 	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.
 * 10) Invert tubes, and tap tubes to beads move to lid region.
 * 11) Poke one hole in the bottom of each tube with hot 21G needle.
 * 12) To do this, heat the needle in a bunsen burner between pokes.
 * 13) 	Place tubes, hole side down, in plastic tubes (from Bell lab). Keep on ice, and move quickly to the next step.
 * 14) 	Spin 5 min at 3000 rpm at 4° to pellet debris. Use the swinging bucket centrifuge in the centrifuge room.
 * 15) 	Recover lysate (the liquid in the tube). Discard the screw-top tube. KEEP ON ICE!
 * 16)      Split lysate into 50 µL aliquots in Epindorf tubes. Keep on ice!
 * 17) 	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.
 * 18) 	1.5 µL Pptase (Calf intestinal Phosphatase)
 * 19) 	22.5 µL pptase inhibitor (see below)
 * 20) 	Incubate all samples at 37 °C for one hour.
 * 21) 	Add 50 µL of 2x sample buffer.
 * 22) 	Boil for 10 minutes.
 * 23) 	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