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

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08/19/10

 * &#x2713; Western blot: remaining Pc-ATF lines for pub. figure
 * &#x2713; 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.

> 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

? min. exposure, BioRad imager (Chemiluminescence, camera)

--> Conclusion:


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