08/19/10
- ✓ Western blot: remaining Pc-ATF lines for pub. figure
- ✓ 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.
Gel 1
1. PageRuler Plus (10 μL)
2,3. KAH129-4 -/+ dox
4,5. KAH156-5 -/+ dox
6,7. KAH130-4 -/+ dox
8,9. KAH158-4 -/+ dox
10. FTRx (-dox)
Gel 2
1. PageRuler Plus (10 μL)
2,3. KAH131-4 -/+ dox
4,5. KAH159-2 -/+ dox
6,7. KAH132-8 -/+ dox
8. FTRx (-dox)
9. dye
10. PageRuler Plus (10 μL)
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Ponceau S stained filters
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> Block: 5% milk/PBST, R.T./1 hr.
> Primary staining: 5% milk/PBST, 4°C/o.n.
- 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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Project name
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