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

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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10/30/10

 * &#x2713; ChIP co-IP optimization: Western blot
 * &#x2713; Cultures: split HepG2 and HEK 23;4;9 (2x 1:5 flasks)
 * &#x2713; Freeze-downs: HepG2 and HEK 23;4;9 (5 tubes, 1 ml ea., from one 75 cm flask)
 * &#x2713; 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.

> 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. > Predicted sizes (using http://www.expasy.ch/tools/pi_tool.html for KAH proteins)
 * 1) donkey α-rabbit-HRP, 1:5000; 5 mL
 * H3K27me3: 15 - 17 kD

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.


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