Update

studies/competition co-IP/protocols/coIP.txt

@@ -14,10 +14,10 @@ Cell harvest:
 5.	Snap freeze in liquid nitrogen Prepare tubes: for concentration determination (BSA dilution 8, 4, 2, 1, 0,5 and 10, 5, 2.5, 1.25, 0.625), 12 tubes for totals mixed with SDS and 12 tubes for -80°C, 12 tubes for the IP reactions, 12 tubes for washing, 12 tubes for eluates, 12 tubes for aliquot of eluates. And HKM washing
 6.	Prepare all buffers: 1% And 0.1% tween buffer, 50% Triton stock
 7.	Prepare 100mM stock of DSP in DMSO (weight 0.016 g in 400 µl DMSO, to 15 mL lysate add 300 µL)
-8.	Resuspend cells (4 Falcons) in 15 (2x7.5 mL) ml HKM (containing: 25 mM EGTA, 200 µg/ml Heparin, 1 mM PMSF, 150 µl Proti, 100 µg/ml CAP; max 8x10^8cells/ml) (10 mM glucose and 20 U/ml hexokinase (Roche) 10 mM ADP (pH 8)
+8.	Resuspend cells (4 Falcons) in 15 (2x7.5 mL) ml HKM (containing: 25 mM EGTA, 200 µg/ml Heparin, 1 mM PMSF, 150 µl Proti, 100 µg/ml CAP, 10 mM glucose and 20 U/ml hexokinase (Roche))
 9.	Thaw the cells carefully
 10.	Add 2 mM DSP (to 7.5 mL add 150 µL)
-11.	Lyse with avestin, two cycles, 900 bar
+11.	Lyse with avestin, two cycles, ~900 bar and add 10 mM ADP (pH 8)
 12.	Crosslink at 4°C for 30 min, (continue lysis of the second 7.5 mL)
 13.	Pool and quench by addition of 100 mM (750 µL of 1 M) Tris pH 8.0, mix
 14.	Remove 60 uL as total and for Bradford assay 

studies/preExperiment/protocols/coIP.txt

-
 HKM Lysis buffer 
 50 mL
 50 mM HEPES pH 8,0 -> 2.5 mL