01/25/10
- ✓ Polycomb-ATF lines: frozen stocks for confluent plates KAH
- ✓ Cultures for Western blot: 12-well dish (~2.5x105/well) for cell lines above
- ✓ Western blot: KAH126-1, -3, -4; KAH127-3, -4; KAH128-2, -6, -8; KAH129-3, -4, -8; KAH132-8; KAH133-6
Western blot
> Bradford assay
--> Calculate amount of protein to load in each lane (6 μL max)
> SDS-PAGE
--> Bring protein vol. up to 6μL w/ dH2O
--> Make 100 mM DTT in 2x loading buffer (200 μL total; > enough for 6 μL/ gel lane)
--> Add 6 μL DTT + buffer to each protein sample
--> Incubate at 100°C/ 5 min.
--> Gel loading:
Gel
- Protein marker
- KAH126-1
- KAH126-3
- KAH126-4
- KAH127-3
- KAH127-4
- KAH128-2
- KAH128-6
- KAH128-8
- KAH129-3
- KAH129-4
- KAH129-8
- KAH132-8
- KAH133-6
- Flp-in T-REx (parent line, no insert)
|
Ponceau S stained filter
|
> Electroblot
--> Run @ 100 V for 1 hr., 15 min. (for large protein transfer)
> Immunostaining
--> Primary: mouse anti-myc, 1:5000 (4°C/ overnight)
--> Secondary: donkey anti-mouse-HRP, 1:10,000 (R.T./ 1 hr.)
Results: Blot showed no signal in any lanes, except for a very faint signal in lane 14. Try again with max. volume of protein.
|