User:Karmella Haynes/Notebook/Polycomb project/2010/08/11

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08/11/10

  • ✓ H3me Reporter line screen: check RFP/YFP (RFP+, but no YFP; check again tomorrow)
  • ✓ Western: KAH130 and 131 clones



Western
> Samples: 22.5 protein + 7.5 4x loading dye
--> 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

1. PageRuler Plus (15 μL)
2,3. KAH130-4 -/+ dox
4,5. KAH130-7 -/+ dox
6,7. KAH130-8 -/+ dox
8,9. KAH130-18 -/+ dox
10. PageRuler Plus

Gel 2
1. PageRuler Plus
2,3. KAH130-19 -/+ dox
4,5. KAH130-22 -/+ dox
6,7. KAH131-4 -/+ dox
8,9. KAH131-8 -/+ dox
10. PageRuler Plus

7/09/10 Ponceau S stain
Ponceau S stained filters

> Block: 5% milk/PBST, R.T./1 hr.
> Primary staining: 5% milk/PBST, 4°C/o.n.
--> rabbit α-myc ab9106, 1:5000, 5 mL


8/12/10
> Secondary staining: 5% milk/PBST, R.T./ 1 hr.
--> donkey α-rabbit, 1:5000, 5 mL
> Predicted sizes (using http://www.expasy.ch/tools/pi_tool.html)

  • KAH130 = 79 kD
  • KAH131 = 79.5 kD


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

--> Conclusion: There is a background band, but unique band appears after dox induction. Keep clones 130-4, 130-8, 130-18, 131-4



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