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

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08/11/10

 * &#x2713; H3me Reporter line screen: check RFP/YFP (RFP+, but no YFP; check again tomorrow)
 * &#x2713; 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.

> 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

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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