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Fabian Ries authoredFabian Ries authored
coIP.txt 3.32 KiB
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, 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 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
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