Buffers/reagents:
Substrate binding buffer (SBB)
50mM Tris pH 7.4, 1mM EDTA, 1M NaCl
Protein binding buffer (PBB)
10mL:
1 protease inhibitor tablet
50mg deacetylase inhibitor, Na butyrate
5mM beta-mercaptoethanol
in (50mM HEPES, 100mM KCl, 10% glycerol, 2.5mM MgCl2, 0.2% NP40)
Reagents:
- streptavidin-conjugated dynabeads (112-05D Invitrogen)
- Spermidine 0.1M from sigma 05292-1ml-F
- BSA 100mg/ml from Sigma, fraction V
- Poly dIdC-dIdC from sigma P4929-5UN-25U. When it arrives, to 25U, add 2.5mL of (20mM Tris HCl pH 8.0, 100mM NaCl), incubate 5min at 45C, chill on ice and aliquot. Store at -20C.
- Na butyrate at 50mM final (Sigma B5887-16). Can store this in ethanol for 1-2months or use it as powder.
- Acetyl-coA Roche 10101893001 10mg. Add in 200uL PBB to make 50mg/mL stock. At -20C, this is stable for 2-3 weeks.
Substrate preparation:
- For closed topology substrate, use these oligos on template pIO2 and PCR out
Dk-gc2: {bi oti nte g}G TTT AGA GGC CCC AAG GGG TTA TGC
Dk-gc31: {BI OTI NTE G}G GCT AGA GTA CTT AAT ACG ACT CAC
- PCR-purify and resuspend in water to get about 100ng/uL.
- For each reaction, use 20uL of dynabeads washed 3x30uL in SBB.
- Add 5-10uL DNA substrate (or 5uL of 0.1M spermidine for negative controls).
- Incubate 1-2hr rocking RT.
- Wash 5x30uL PBB, resuspend in 55uL PBB.
- add BSA (5uL) and dIdC (5uL), incubate 30min-1hr RT while preparing cell extracts.
- add Acetyl-CoA (5uL) just before starting the reaction.
Cell preparation
- In the morning put a single colony in 5 mL YPD or 5uL of liquid culture in 5mL YPD. Let grow all day. After 8-9 hours the OD600 should be ~1
- Dilute appropriately into 500 mL YPD (typically 0.5-1mL). Grow O/N to get OD600=0.5 in the morning
- If cells overgrew: OD600>1 discard cells and start over; OD600<1 dilute cells to 0.1 and grow to OD600=0.5
- Add HU (1.5g per 500mL culture, 0.2M final) at OD600=0.5. Incubate for 3h at 30C.
- Spin cells down, 3000 rpm, 3 min.
- Resuspend in 5 ml sterile water
- Split into 6 screw cap tubes
- Spin 10 sec, 14K rpm
- Remove sup completely by vacuum.
- Freeze tubes in liquid N2
- Keep pellets in -80C
NOTES:
- Freezing in N2 and keeping the pellets in -80C is critical to maintain activity. Activity is severely reduced if cells are frozen by any other method and kept at -20C.
- Starting from mid-log starter is important
- Visualize cells after HU arrest to confirm synchronization
Extract preparation
- Thaw pellets on ice ~5 min, add 350 uL chilled buffer
- Add 1 scoop of glass beads. (glass beads should be stored @-20C)
- Break in beat beater set on full-power 30 sec beating, 1min pause x 3 times
- Punch tube in the bottom middle and 1-2 more time on the bottom sides
- Spin into 2 mL tube 1K rpm, 2 min, 4C
- Put sup. in a new 1.5 ml tube.
- Spin 12K rpm, 15min, 4C
Koshland lab bead beater
NOTES:
- The setting is for the 5417 centrifuge.
- The protein amount of a typical extract should be 15-20 mg/ml (by Bradford assay)
Reaction:
total 100uL to be incubated 30min 30C
70uL reaction mix (55uL buffer+beads, 5uL Acetyl CoA, 5uL BSA, 5uL dIdC)
+30uL yeast extract
salt washes (0.1M or 0.5M KCl in PBB).
Wash in 100uL volume. Do 1x LS, 2xLS or HS, 1xLS wash.
Resuspend in 30uL 2x Sample buffer
NOTES:
- According to the kinetics 30 min are required to achieve steady-state
- Do not pipet beads up and down
- Do the washing as fast as you can, one tube at a time. Avoid keeping the beads without buffer.
- The freshness of Acetyl CoA is important. Store solution in -20C freezer up to 2 weeks. Discard afterwards.
SDS-PAGE and protein transfer
- Leave at least one lane empty between input and samples. Load juice in the empty lane. Otherwise input will diffuse into it. Alternatively, check inputs on a separate gel.
- Either wet transfer 40V, O/N OR Semi-Dry (BioRad Turbo) 30min standard program (25V-1A)
Western blot
- Washes done in PBS-T 0.1% tween 20
- Blocks and antibody dilutions in 5% milk in PBS-T
- Block 10-30min
- Use Vinny’s affinity purified anti-Mcd1 (overnight)
- Secondary Ab 30min-1hr, RT
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