User:Karmella Haynes/Notebook/Polycomb project/2010/08/27

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08/27/10

 * &#x2713; Western blot: ChIP/ Co-IP
 * &#x2713; 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.

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

> Primary staining: 5% BSA/PBST, 4°C/o.n.
 * 1) rabbit α-DsRed 632496, 1:1000, 5 mL
 * 2) rabbit α-H3K27me3 07-449, 1:500, 5 mL

8/28/10 > 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-rabbit-HRP, mouse α-GAPDH-HRP 1:5000; 5 mL
 * 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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