User:Karmella Haynes/Notebook/Polycomb project/2010/10/30

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10/30/10

  • ✓ ChIP co-IP optimization: Western blot
  • ✓ Cultures: split HepG2 and HEK 23;4;9 (2x 1:5 flasks)
  • ✓ Freeze-downs: HepG2 and HEK 23;4;9 (5 tubes, 1 ml ea., from one 75 cm flask)
  • ✓ Transfection prep: set up 2 6-well plates for HepG2 (0.5 mL 1:10 dilution per well



Western: ChIP co-IP
> Optimization IP for αH3K27me3
> IP samples from 10/29/10; 60 μL each, ready to load
> Prep other samples: 22.5 protein + 7.5 4x loading dye
--> 4x loading dye (Invitrogen) w/ freshly added DTT (200 mM final)
> Use 10-well gel (loading volume = 25 μL)
> Electroblot: 1 hr. 30 min.

Gel 1

1. Benchmark ladder (10 μL)
2. KAH126-1 dx input
3. KAH126-1 dx + α-H3K27me3 Sup
4. " W1 (Low Salt sup)
5. " W2 (High Salt sup)
6. " W3 (LiCl wash sup)
7. " W4 (TE sup)
8. " IP
9. ladder
10. 1x dye

Gel 2
1. Benchmark ladder (10 μL)
2. KAH126-1 dx input
3. KAH126-1 dx + α-rabbit IgG Sup
4. " W1 (Low Salt sup)
5. " W2 (High Salt sup)
6. " W3 (LiCl wash sup)
7. " W4 (TE sup)
8. " IP
9. ladder
10. 1x dye

10/30/10 Ponceau S stain
Ponceau S stained filters

> Block: 5% milk/PBST, RT/ > 1 hr.

> Primary staining: 5% BSA/PBST, 4°C/o.n.

  1. rabbit α-H3K27me3 07-449, 1:1000, 3.0 mL


10/31/10
> Secondary staining: 5% milk/PBST, R.T./ 1 hr.

  1. donkey α-rabbit-HRP, 1:5000; 5 mL

> Predicted sizes (using http://www.expasy.ch/tools/pi_tool.html for KAH proteins)

  • H3K27me3: 15 - 17 kD


Western blot 10/31/10
Conclusions: Got rid of non-specific binding, but don't see much signal in the H3K27me3 IP lane. Try again with experimental samples and use only 1x washes.