From e954cf2bea5b5058d713cdb5fe499752eb4a6cb3 Mon Sep 17 00:00:00 2001
From: riesf <riesf@rhrk.uni-kl.de>
Date: Thu, 16 Jun 2022 12:16:46 +0200
Subject: [PATCH] Update

---
 studies/.DS_Store                            | Bin 6148 -> 6148 bytes
 studies/competition co-IP/protocols/coIP.txt |   4 ++--
 studies/preExperiment/protocols/coIP.txt     |   1 -
 3 files changed, 2 insertions(+), 3 deletions(-)

diff --git a/studies/.DS_Store b/studies/.DS_Store
index 7dd4770b6613bc75059485514a31eef53ad6fdd3..28f777e6d836ee2c5e30d692d2a68a8e39e109a1 100644
GIT binary patch
delta 211
zcmZoMXfc=|#>B!ku~2NHo}wrV0|Nsi1A_nqLn=cFLnavKZ#HCJ&TI^l5@bkb$Y;o9
zC_t8G$YW3di|aCYG6W=*7Z)Vu<R<}DpO|dM)W9WPU2SNjqhMlSKKUS%EaQyH_nG94
zXA);vS#VKaPJUiG$W@d7Gs$h%V%o^GnVo~51L$O+v%WJ=<`;3~0J@k7sDxp2gvc6Z
E0Gp&O@c;k-

delta 89
zcmZoMXfc=|#>B)qu~2NHo}wrd0|Nsi1A_nqLj^+tLn=cNLncG+#=_-{lMO^zChM_D
qZw_PK$h5IRhj}wQ2R{c;<7PpQ@640=MI1SRT0weQHb;o8VFmzaT@v>I

diff --git a/studies/competition co-IP/protocols/coIP.txt b/studies/competition co-IP/protocols/coIP.txt
index cec1ca75e..a94dd4858 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 f1cea89fe..73017f8f7 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
-- 
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