05/18/10
- ✓ Western blot: optimize ab's for ChIP
Western blot
> SDS-PAGE
--> 2x 10-well tris-glycine (4-20%) pre-cast gels
--> Samples (25 μL total vol./ well, dilute protein w/ RIPA):
- PageRuler Plus pre-stained ladder: 15 μL
- U2OS, 12.5 μL, ~ 29 μg protein
- U2OS, 6.0 μL, ~ 14 μg
- U2OS, 3.0 μL, ~ 7 μg
- U2OS, 1.5 μL, ~ 3.5 μg
--> Make 100 mM DTT in 2x loading buffer (> enough for 12.5 μL/ gel lane)
--> Add 12.5 μL DTT + buffer to each protein sample
--> Incubate at 100°C/ 5 min.
--> Run gel @ 120 V until dye front reaches the bottom
> Electroblot
--> Run @ 100 V for 1 hr. (blot 1, histones), and 1.5 hr (blot 2, large proteins)
--> Cut each filter in half & trim (4 sets of samples total))
| Gel loading
- PageRuler Plus pre-stained ladder
1. U2OS, 29 μg
2. U2OS, 14 μg
3. U2OS, 7 μg
4. U2OS, 3.5 μg
|
Ponceau S stained filters
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> Immunostaining
--> Block 4°C/ overnight; continue Wed. through Fri.
5/19/10
--> Primaries (4°C/ overnight):
- rabbit anti-H3 (ab1791), 1:5000, 5 mL
- rabbit anti-H3K27me3 (07-449), 1:1000, 5 mL
- mouse anti-PolII (ab817), 1:1000, 5 mL
- mouse anti-BMI1 (ab14389), 1:1000, 5 mL
5/20/10
--> Secondary:
1,2. donkey anti-rabbit IgG-HRP, 1:5,000 (R.T./ 1 hr.)
3,4. donkey anti-mouse IgG-HRP, 1:5,000 (R.T./ 1 hr.)
(Molecular weights computed at http://www.expasy.ch/tools/pi_tool.html)
Lanes
- PageRuler Plus pre-stained ladder
- U2OS, 29 mg
- U2OS, 14 mg
- U2OS, 7 mg
- U2OS, 3.5 mg
Signals
- Blot 1, H3 = 15 kD
- Blot 2, H3K27me3 = > 15 kD
- Blot 3, PolII = 217 kD
- Blot 4, BMI1 = 42 kD
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Western blot 5/19/10
## min. exposure
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> Coclusions:
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Project name
Main project page
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