Koshland Web Site/Methods
In Vitro Cohesin Loading Assay

Substrate binding buffer (SBB)
50mM Tris pH 7.4, 1mM EDTA, 1M NaCl
Protein binding buffer (PBB)
            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)
-            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
- 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

  1. 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
  2. Dilute appropriately into 500 mL YPD (typically 0.5-1mL). Grow O/N to get OD600=0.5 in the morning
  3. If cells overgrew:  OD600>1 discard cells and start over; OD600<1 dilute cells to 0.1 and grow to OD600=0.5
  4. Add HU (1.5g per 500mL culture, 0.2M final) at OD600=0.5. Incubate for 3h at 30C.
  5. Spin cells down, 3000 rpm, 3 min.
  6. Resuspend in 5 ml sterile water
  7. Split into 6 screw cap tubes
  8. Spin 10 sec, 14K rpm
  9. Remove sup completely by vacuum.
  10. Freeze tubes in liquid N2
  11. Keep pellets in -80C   


  1. 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.
  2. Starting from mid-log starter is important
  3. Visualize cells after HU arrest to confirm synchronization

Extract preparation

  1. Thaw pellets on ice ~5 min, add 350 uL chilled buffer
  2. Add 1 scoop of glass beads. (glass beads should be stored @-20C)
  3. Break in beat beater set on full-power 30 sec beating, 1min pause x 3 times
  4. Punch tube in the bottom middle and 1-2 more time on the bottom sides
  5. Spin into 2 mL tube 1K rpm, 2 min, 4C
  6. Put sup. in a new 1.5 ml tube.
  7. Spin 12K rpm, 15min, 4C

Koshland lab bead beater


  1. The setting is for the 5417 centrifuge.
  2. The protein amount of a typical extract should be 15-20 mg/ml (by Bradford assay)

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


  1. According to the kinetics 30 min are required to achieve steady-state
  2. Do not pipet beads up and down
  3. Do the washing as fast as you can, one tube at a time. Avoid keeping the beads without buffer.
  4. The freshness of Acetyl CoA is important. Store solution in -20C freezer up to 2 weeks. Discard afterwards.

SDS-PAGE and protein transfer

  1. 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.
  2. Either wet transfer 40V, O/N OR Semi-Dry (BioRad Turbo) 30min standard program (25V-1A)

Western blot

  1. Washes done in PBS-T 0.1% tween 20
  2. Blocks and antibody dilutions in 5% milk in PBS-T
  3. Block 10-30min
  4. Use Vinny’s affinity purified anti-Mcd1 (overnight)
  5. Secondary Ab 30min-1hr, RT
Comments: prepared by Itay Onn and Gamze Camdere
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