User:Karmella Haynes/Notebook/Polycomb project/2010/08/19

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08/19/10

  • ✓ Western blot: remaining Pc-ATF lines for pub. figure
  • ✓ Cell culture: split confluent lines



Western
> Samples: 22.5 protein + 7.5 4x loading dye
--> 4x loading dye (Invitrogen) w/ freshly added DTT (100 mM final)
> Use 10-well gel (loading volume = 25 μL)
> Electroblot: 1 hr. 15 min.

Gel 1

1. PageRuler Plus (10 μL)
2,3. KAH129-4 -/+ dox
4,5. KAH156-5 -/+ dox
6,7. KAH130-4 -/+ dox
8,9. KAH158-4 -/+ dox
10. FTRx (-dox)

Gel 2
1. PageRuler Plus (10 μL)
2,3. KAH131-4 -/+ dox
4,5. KAH159-2 -/+ dox
6,7. KAH132-8 -/+ dox
8. FTRx (-dox)
9. dye
10. PageRuler Plus (10 μL)

File:KAH 081910 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-DsRed 632496, 1:2000, 5 mL


8/20/10
> 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)

  • KAH129-4 = 43 kD
  • KAH156-5 = 37.5 kD
  • KAH130-4 = 79 kD
  • KAH158-4 = 73.5 kD
  • KAH131-4 = 79.5 kD
  • KAH159-2 = 74 kD


File:KAH 082010 WB1.tif
? min. exposure, BioRad imager (Chemiluminescence, camera)

--> Conclusion: