diff --git a/studies/.DS_Store b/studies/.DS_Store index 7dd4770b6613bc75059485514a31eef53ad6fdd3..28f777e6d836ee2c5e30d692d2a68a8e39e109a1 100644 Binary files a/studies/.DS_Store and b/studies/.DS_Store differ diff --git a/studies/competition co-IP/protocols/coIP.txt b/studies/competition co-IP/protocols/coIP.txt index cec1ca75e0b02aef2ca5643edd901d8d647d32a2..a94dd4858b4b612f6a388bb05866a084c96cd728 100644 --- a/studies/competition co-IP/protocols/coIP.txt +++ b/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 diff --git a/studies/preExperiment/protocols/coIP.txt b/studies/preExperiment/protocols/coIP.txt index f1cea89fe6c6eeb8075141f3323374483aaa0cf2..73017f8f77a59bc088660f2d0b7f8331985465d7 100644 --- a/studies/preExperiment/protocols/coIP.txt +++ b/studies/preExperiment/protocols/coIP.txt @@ -1,4 +1,3 @@ - HKM Lysis buffer 50 mL 50 mM HEPES pH 8,0 -> 2.5 mL