User:Karmella Haynes/Notebook/Polycomb project/2010/08/13

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08/13/10

  • ✓ Western: Pc-ATF lines -/+ dox (for pub. figure)
  • ✓ Microscopy: KAH130 and 131 lines; RFP vs. DAPI
  • ✓ Senescence assay (replicate #3): 6-well plate KAH126-1, 126-3, 154-2, 132-8 -/+dox; FTRx +DMSO, rotenone, dox, u.t.
  • ✓ Cell cultures: split confluent plates



Western
> Samples: 22.5 protein + 7.5 4x loading dye (enough for 2 wells each)
--> 4x loading dye (Invitrogen) w/ freshly added DTT (100 mM final)
> Use 15-well gel (loading volume = 12 μL)
> Electroblot: 1 hr. 15 min.

Gels 1 & 2

1. PageRuler Plus (15 μL)
2,3. KAH126-1 -/+ dox
4,5. KAH154-2 -/+ dox
6,7. KAH128-8.3 -/+ dox
8,9. KAH157-1 -/+ dox
10,11. KAH129-4 -/+ dox
12,13. KAH156-5 -/+ dox
14. FTRx (-dox)
10. PageRuler Plus

File:KAH 081310 ps1.tif
Ponceau S stained filters

> Block: 5% milk/PBST, R.T./1 hr.

> Primary staining: 5% milk/PBST, 4°C/o.n.

  1. rabbit α-myc ab9106, 1:5000, 5 mL
  2. rabbit α-DsRed 632496, 1:4000, 5 mL


8/14/10
> Secondary staining: 5% milk/PBST, R.T./ 1 hr.
--> donkey α-rabbit-HRP, 1:5000; mouse α-GAPDH-HRP, 1:5000, 5 mL
> Predicted sizes (using http://www.expasy.ch/tools/pi_tool.html)

  • KAH126-1 = 43 kD
  • KAH154-2 = 37.5 kD
  • KAH128-8.3 = 43 kD
  • KAH157-1 = 37.5 kD
  • KAH129-4 = 43 kD
  • KAH156-5 = 37.5 kD


Western blot 8/12/10
1 min. exposure, BioRad imager (Chemiluminescence, camera)

--> Conclusion: Used expired gels (April 2010). Image looks very bad. GAPDH no visible in all wells. Repeat with 10-well gels.