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

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08/13/10

 * &#x2713; Western: Pc-ATF lines -/+ dox (for pub. figure)
 * &#x2713; Microscopy: KAH130 and 131 lines; RFP vs. DAPI
 * &#x2713; Senescence assay (replicate #3): 6-well plate KAH126-1, 126-3, 154-2, 132-8 -/+dox; FTRx +DMSO, rotenone, dox, u.t.
 * &#x2713; 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.

> 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

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.


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