User:Karmella Haynes/Notebook/Polycomb project/2010/06/30

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06/30/10

  • ✓ ChIP: Western for myc & control IP samples
  • ✓ Western: protein preps for Pc-ATF lines 128-8.3, 127-4 (130-7 showed no RFP in dox sample, discarded)
  • ✓ Transfection (prep): refresh ab-free medium for FTRx H3me3 colony picks (reporter test: transfect w/ Gal4-VP64 tomorrow)



ChIP: wash, elute, Western blot
> Wash & elute IP (Keep everything on ice)
--> Prepare washing buffer (6 mL complete sonication buffer plus PLAAC, PMSF, detergent, salts, etc.).
--> Spin down beads at 3000 rpm/ 4°C
--> Save 50 μL supernatant. Discard remaining sup. Wash beads in 500 μL wash buffer.
--> Save 50 μL wash 1 sup. Discard remaining sup. Wash beads in 500 μL wash buffer.
--> Save 50 μL wash 2 sup. Discard remaining sup. Wash beads in 500 μL wash buffer.
--> Discard sup. and wash once more (4 washes total).
--> Add 10 μL 4x loading dye to each pellet. Heat at 100°C/ 5 min., vortex, heat again.
--> Clear supernatant by spinning at 4000 rpm/ 2 min. Transfer 10 μL sup to new tube (for Western).


> Western blot loading
> Sup, wash, & input samples: 18.75 protein + 6.25 4x buffer
> Use 4x loading dye (Invitrogen) w/ BME (400 μL loading dye + 40 μL BME)
> Use 10-well gel (loading volume = 25 μL)
> Electroblot: 1 hr. 15 min.

Gel
  1. PageRuler Plus (10 μL)
  2. myc IP sup
  3. myc IP wash 1
  4. myc IP wash 2
  5. myc IP
  6. bead IP sup
  7. bead IP wash 1
  8. bead IP wash 2
  9. bead IP
  10. 132-8 input
File:KAH 063010 ps1.tif
Ponceau S stained filter

> Block: 5% milk/PBST, R.T./1 hr.
> Primary staining: 5% BSA/PBST, 4°C/o.n.
--> rabbit α-myc ab9106, 1:1000, 5 mL


7/01/10
> Secondary staining: 5% milk/PBST, R.T./ 1 hr.
--> donkey α-rabbit, 1:5000, 5 mL