2 Days Before--Start 5 mL overnight from single colony.

1 Day Before--Subculture overnight into110 mL (large flask for aeration). Dilute so ~2×10⁷cells/mL at convenient time on Day 1.

Reserve Sorvall centrifuge (SS34, 4℃). Cool large rotor (TMA-3E) for TOMY centrifuge.

Day 1   Crosslinking Cells

1. When culture is ready (usually I aim for OD₆₀₀ = 1.2; I've used up to 1.5), add formaldehyde directly to the medium--1% final concentration. Calculation: VOL of culture/36 = mL 37% H₂C=O. Fix at room temperature with gentle swirling (at least occasionally). Fixation time from 10 min to 2 hours
2. Transfer cells to 50 mL conical tubes and spin down in clinical centrifuge.
3. Spheroplast cells. Volumes are for 100 mL of cells.
4. Resuspend cells in a total of 5 mL 0.1 M TRIS (pH 9.4), 10 mM DTT (freshly prepared), pool, and transfer to Nalgene Oak Ridge tube. Place on ice for 15-20 minutes.
5. Spin down in Sorvall--5 K, 5 minutes, 4℃. Drain.
6. Resuspend cells in 5 mL HEPES/sorb to wash. Spin down (5 K, S minutes, 4℃). Drain.
7. Resuspend in 5 mL HEPES/sorb with 0.5 mM PMSF. Add 30-40 mL 1 mg/mL oxalyticase (can also use 2 mg zymolyase here, as preferred). Incubate at 30℃ for 30 minutes with gentle agitation
8. Add l0 mL PIPES/sorb with 0.5 mM PMSF. Spin down immediately--5 K, 5 minutes, 4℃. Drain.
9. Wash spheroplasts (3x). All manipulations on ice.
10. Gently resuspend cell pellet in 5 mL cold PBS with 0.5 mM PMSF. Spin down @ 5K, 5 minutes, 4℃. Drain.
11. Gently resuspend cell pellet in 5 mL cold Triton X/HEPES with 0.5 mM PMSF, 0.8 ug/mL pepstatin A, and 0.6 ug/mL leupeptin. Spin down @ 7 K, 7 minutes, 4℃. Drain.
12. Gently resuspend cell pellet in 5 mL cold NaCl/HEPES with 0.5 mM PMSF, 0.8 ug/mL pepstatin A, and 0.6 ug/mL leupeptin. Spin down @ 7 K, 7 minutes, 4℃. Drain.
13. Sonicate spheroplasts
14. Resuspend cell pellet in 1 mL SDS lysis buffer with 1 mM PMSF, 0.8 ug/mL pepstatin A, and 0.6 ug/mL leupeptin. Transfer to 2 Eppendorf tubes (divide equally).
15. Sonicate suspension on ice for 10 second intervals, with at least 5 minutes on ice in between, until
    DNA is 100-2000 bp range (avg 400-500, Branson Sonifier 250: constant output @ 15-20% power, 6 pulses, 10 sec each.
16. Spin in microfuge at maximum speed, 10 minutes, 4℃-10℃ (TOMY @ 15K ~18,000 x gmax)
17. Chromatin solution
18. Transfer sup (maybe cloudy from the SDS) to 15 mL Falcon snapcap tube on ice (expect ~1.1mL).
19. Add 10 mL IP Dilution Buffer with 1 mM PMSF, 0.8 ug/mL pepstatin A, and 0.6 ug/mL leupeptin (10 mL ~9 volumes SDS lysate; final [SDS] ~ 0.1%). Let sit on ice awhile.
20. Spin in TOMY @ 10K (~8,400 x gmax), 10 minutes, 4℃-10℃. Decant sup into 15 mL conical tube. Place on ice. This is the chromatin solution.
21. Set up immunoprecipitations
22. Aliquot chromatin solution to Eppendorf tube (I use 1.5 mL per IP; ~15-20 O.D.₆₀₀ equivalents).
23. Add appropriate volume of primary antibody. Incubate overnight 4℃ on Nutator. I usually set up duplicate IPs.
    Note: Always do NO Ab control. Also, if adding competitor antigen, do so several minutes before adding Ab. It may be desirable to denature antigen in SDS first. In this case keep track of the additional SDS and supplement companion IPs to the same final [SDS].

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