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

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11/15/10

 * &#x2713; Order: H3 (#12-565) and H3K27me3-biotin (#12-404) peptides and streptavidin-beads from Millipore
 * &#x2713; 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): > 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.
 * 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

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. > Predicted sizes (using http://www.expasy.ch/tools/pi_tool.html for KAH proteins)
 * 1) donkey α-rabbit-HRP, 1:5000; 5 mL
 * 2) donkey α-mouse-HRP, 1:5000; 5 mL
 * H3K27me3: 15 - 17 kD
 * KAH126-1: 43 kD
 * KAH132-8: 35 kD
 * KAH130-4: 79 kD

Conclusions:


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