User:Karmella Haynes/Notebook/Polycomb project/2010/11/15

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11/15/10

  • ✓ Order: H3 (#12-565) and H3K27me3-biotin (#12-404) peptides and streptavidin-beads from Millipore
  • ✓ Western blot: ChIP optimization trial from 11/02/10


Western blot: ChIP optimization
> Use IP samples from 11/02/10
> Run 2 gels
> Samples (all chromatin is CH2O single x-linked):

  1. PageRuler Plus (10 uL)
  2. 126-1 input
  3. 126-1 H3K27me3 (07-449) IP
  4. 126-1 rabbit IgG IP
  5. 132-8 input
  6. 132-8 H3K27me3 (07-449) IP
  7. 132-8 rabbit IgG IP
  8. 130-4 input
  9. 130-4 H3K27me3 (07-449) IP
  10. 130-4 rabbit IgG IP

> Loading: 25 uL total; IP samples ready to load; Input = 22.5 protein + 7.5 4x dye(+200 mM DTT)
> Heat all samples at 100°C/ 5 min.
> Electro-blot 2 hrs.

11/15/10 Ponceau S stain
Ponceau S stained filters
Note: accidentally blotted this ~25 min longer than intended, but transfer looks great

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

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

  1. rabbit α-H3K27me3 07-449, 1:1000, 5.0 mL
  2. mouse α-myc , 1:1000, 5.0 mL


11/16/10
> Secondary staining: 5% milk/PBST, R.T./ 1 hr.

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

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

  • H3K27me3: 15 - 17 kD
  • KAH126-1: 43 kD
  • KAH132-8: 35 kD
  • KAH130-4: 79 kD


Western blot 11/16/10
Conclusions:





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