08/27/10
- ✓ Western blot: ChIP/ Co-IP
- ✓ Microscopy: secondary staining and mounting (see 8/26/10)
Western blot: ChIP/ Co-IP
> IP samples from 8/20/10; already prepped for loading (2 wells worth)
> Prep input and sup 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.
Gels 1 & 2
1. PageRuler Plus (10 μL)
2. KAH126-1 input
3. KAH126-1 α-myc sup
4. KAH126-1 α-myc IP
5. KAH126-1 α-IgG IP (neg ctrl)
6. KAH132-8 input
7. KAH132-8 α-myc sup
8. KAH132-8 α-myc IP
9. KAH132-8 α-IgG IP (neg ctrl)
10. PageRuler Plus (10 μL)
Note: In gel #2, samples misloaded as:
1, 4, 5, 2, 3, 6...10
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Ponceau S stained filters
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> Block: 5% milk/PBST, R.T./1 hr.
> Primary staining: 5% BSA/PBST, 4°C/o.n.
- rabbit α-DsRed 632496, 1:1000, 5 mL
- rabbit α-H3K27me3 07-449, 1:500, 5 mL
8/28/10
> Secondary staining: 5% milk/PBST, R.T./ 1 hr.
- donkey α-rabbit-HRP, 1:5000; 5 mL
- donkey α-rabbit-HRP, mouse α-GAPDH-HRP 1:5000; 5 mL
> Predicted sizes (using http://www.expasy.ch/tools/pi_tool.html for KAH proteins)
- KAH126-1: 43 kD
- KAH132-8: 35 kD
- H3K27me3: 15 - 17 kD
Conclusions:
- KAH126 pull-down was successful and specific for α-myc beads (top); no signal for KAH132 (?)
- Both KAH126 and 132 appear to Co-IP with 28 kD band in H3K27me3 staining; no idea what this is (di-nucleosomes?)
- Perhaps DMA/ formaldehyde is too much crosslinking, given that Pc-ATFs are over-expressed and concentrated in the nucleus
- Try new preps, using formaldehyde x-linking only
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Project name
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