Update

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

-Competition co-IP:
-1.
\ No newline at end of file
+
+HKM Lysis buffer 
+50 mL
+50 mM HEPES pH 8,0 -> 2.5 mL
+25 mM KCl -> 0.625 mL
+25 mM MgCl2 -> 1.25 mL
+100 µg/ml CAP -> 50 µL
+	 
+Cell harvest:
+1.	Grow 3x 2000 ml of a cw15 (2) strain to a density of 4-5 x 10^6 cell/ml (150 ml cells = 7.5 x10^8 cells per IP)
+2.	Add 100µg/ml CAP prior to the first 1 L, pour the culture onto ice cubes (chilled at -80°C)
+3.	Spin the cells min at 3500xg and 4°C for 2 min
+4.	Wash the cells in 100 ml HKM-CAP, pool in two 50 ml Falcon 
+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)
+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
+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 
+15.	Add 1% Triton incubate 5 min on ice with some mixing 
+16.	Centrifuge 15 min at 20,000 g and 4°C in 2 mL tubes (in the meanwhile wash the affinity beads. Wash slurry 2x with HKM, reconstitute in 650 uL 
+17.	Recover the supernatant, this is your input lysate
+18.	Ensure that your lysate is homogenous
+19.	Determine the protein concentration with Bradford from the total aliquot (dilute 10, 5, 2.5, 1.25, 0.625 µl lysate into 10 µl HKM-Lysis buffer; and add 800 µl 1xBradford reagent; incubate 5 min at RT and measure at 595nm; make a fresh Bradford series with BSA (8,4,2,1,0.5 µg).
+20.	Prepare 3x6 tubes for the different reactions, with 1200 µl lysate each or less if some is lost somewhere
+21.	Calculate how much of your 15N bait protein you need to add (0.25:1, 0.5:1, 1:1, 2.5:1, 5:1, 10:1 15N:14N)
+22.	After the addition of the 15N protein take a 25 µl aliquot (freeze in -80°C)
+IP
+23.	To each reaction add 100µl slurry, let the beads bind the epitopes for 1h at 4°C
+24.	Wash, spin for 60 sec. at 1000 g and 4°C
+25.	Transfer 50 µl of supernatant to a new tube and label it “flow-through”, and freeze at -80°C. Discard remaining supernatant.
+26.	Now wash (The washing should be done by a end ever end shaker for 2 min at 4°C) the beads 3 X with 750 µL HKM (1% Tween-20).
+27.	Resuspend the beads with 500µl HKM (0.1% Tween-20) and transfer to a new tube, again wash old tube carefully with 500µl and add to new tube 
+28.	spin again and wash two more times with 750µl HKM and 0.1% Tween
+29.	spin down, remove the supernatant carefully, add 100 µl 2x SDS-buffer without DTT
+30.	boil all samples 1 min at 95°C
+31.	Take the eluate from the beads with a 100 µl Hamilton pipette (ensure good washing of the pipet, SDS running buffer, H2O, H2O)
+32.	Add 50mM DTT and boil again 5 min to cleave the crosslinker 
+33.	Take 10 µL for western, freeze the rest at -20°C

studies/preExperiment/protocols/coIP.txt

-Competition co-IP:
-1.
\ No newline at end of file
+
+HKM Lysis buffer 
+50 mL
+50 mM HEPES pH 8,0 -> 2.5 mL
+25 mM KCl -> 0.625 mL
+25 mM MgCl2 -> 1.25 mL
+100 µg/ml CAP -> 50 µL
+	 
+Cell harvest:
+1.	Grow 3x 2000 ml of a cw15 (2) strain to a density of 4-5 x 10^6 cell/ml (150 ml cells = 7.5 x10^8 cells per IP)
+2.	Add 100µg/ml CAP prior to the first 1 L, pour the culture through wet ice.	Spin the cells min at 3500xg and 4°C for 2 min
+4.	Wash the cells in 100 ml HKM-CAP, pool in two 50 ml Falcon 
+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)	
+9.	Thaw the cells carefully
+10.	Add 2 mM DSP (to 7.5 mL add 150 µL)
+11.	Potter-Elvehjem homogenizer, 20 strikes
+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 
+15.	Add 1% Triton incubate 5 min on ice with some mixing 
+16.	Centrifuge 15 min at 20,000 g and 4°C in 2 mL tubes (in the meanwhile wash the affinity beads. Wash slurry 2x with HKM, reconstitute in 650 uL 
+17.	Recover the supernatant, this is your input lysate
+18.	Ensure that your lysate is homogenous
+19.	Determine the protein concentration with Bradford from the total aliquot (dilute 10, 5, 2.5, 1.25, 0.625 µl lysate into 10 µl HKM-Lysis buffer; and add 800 µl 1xBradford reagent; incubate 5 min at RT and measure at 595nm; make a fresh Bradford series with BSA (8,4,2,1,0.5 µg).
+20.	Prepare 2x6 tubes for the different reactions, with 1200 µl lysate each or less if some is lost somewhere
+21.	Calculate how much of your 15N bait protein you need to add (0.05:1, 0.25:1, 0.5:1, 1:1, 5:1, 10:1 15N:14N)
+22.	After the addition of the 15N protein take a 25 µl aliquot (freeze in -80°C)
+IP
+23.	To each reaction add 100µl slurry, let the beads bind the epitopes for 1h at 4°C
+24.	Wash, spin for 60 sec. at 1000 g and 4°C
+25.	Transfer 50 µl of supernatant to a new tube and label it “flow-through”, and freeze at -80°C. Discard remaining supernatant.
+26.	Now wash (The washing should be done by a end ever end shaker for 2 min at 4°C) the beads 3 X with 750 µL HKM (1% Tween-20).
+27.	Resuspend the beads with 500µl HKM (0.1% Tween-20) and transfer to a new tube, again wash old tube carefully with 500µl and add to new tube 
+28.	spin again and wash one more times with 750µl HKM and 0.1% Tween, and two times without detergent
+29.	spin down, remove the supernatant carefully, add 100 µl 2%SDS in 40 mM ammonium bicarbonate
+30.	boil all samples 1 min at 95°C
+31.	Take the eluate from the beads with a 100 µl Hamilton pipette (ensure good washing of the pipet, 2% SDS, H2O, H2O)
+32.	Add 50mM DTT and boil again 5 min to cleave the crosslinker 
+33.	Take 10 µL for western, freeze the rest at -20°C