User:Karmella Haynes/Notebook/Polycomb project/2010/07/16

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07/16/10

 * &#x2713; Western blot: Pc-ATF set (for pub.); -/+ rotenone p16INK (CDKN2a)

Western > Western blot loading --> 10-well Samples: 22.5 protein + 7.5 4x loading dye --> 15-well Samples: 15.0 protein + 5.0 4x loading dye --> Use 4x loading dye (Invitrogen) w/ BME (400 μL loading dye + 40 μL BME) > Electroblot: 1 hr. 15 min.

> Block: 5% milk/PBST, R.T./1 hr. > Primary staining: 4°C/o.n. --> Filter 1a: mouse α-p16 ab16880, 1:100, 2 mL 5% BSA/PBST --> Filter 1b: mouse α-p16 ab16880, 1:50, 2 mL 5% BSA/PBST --> Filter 2: rabbit α-myc ab9106, 1:1000, 5 mL 5% milk/PBST

7/17/10 > Secondary staining: 5% milk/PBST, R.T./ 1 hr. --> Filters 1a, 1b: donkey α-mouse, 1:5000 --> Predicted size: 16 kD (http://www.uniprot.org/uniprot/P42771)

--> Filter 2: donkey α-rabbit, 1:5000, 5 mL; mouse α-GAPDH-HRP, 1:5000 --> 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
 * KAH132-8 = 35 kD

7/17/10 -- exposure, BioRad imager (Chemiluminescence, camera)

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