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

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07/16/10

  • ✓ 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.

Gel 1

1. PageRuler Plus (15 μL)
2. FTRx, 10 cm plate
3. FTRx, 10 cm plate, +0.2 μg/mL rotenone
4. FTRx, 6-well plate
5. FTRx, 6-well plate, +0.2 μg/mL rotenone
6-10. Duplicate of 1-5
--> Cut in half for immunostaining

Gel 2
1. PageRuler Plus (10 μL)
2,3. KAH126-1 -/+ dox (6/15/10)
4,5. KAH154-2 -/+ dox (5/21/10)
6,7. KAH128-8.3 -/+ dox (6/30/10)
8,9. KAH157-1 -/+ dox (5/21/10)
10,11. KAH129-4 -/+ dox (7/02/10)
12,13. KAH156-5 -/+ dox (5/21/10)
14,15. KAH132-8 -/+ dox (6/15/10)

File:KAH 071610 ps1.tif
Ponceau S stained filters


> 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 Western blot 7/11/10
-- exposure, BioRad imager (Chemiluminescence, camera)

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