User:Karmella Haynes/Notebook/Polycomb project/2010/10/28

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10/28/10
--> Note: HEK 23;4;9 has a Tet repressor, so Pc-ATF needs dox to be expressed! Add dox and assay RFP/ luc when cells are confluent
 * &#x2713; HEK Gal4EED + Pc-ATF transf.: RFP expression is low, refresh medium and check again tomorrow
 * &#x2713; ChIP: overnight IP for optimization assay

ChIP co-IP optimization > Not much luck with endogenous protein IP using previous simplified protocol (See 10/06/10) > Try protocol from Casey: --> Bind antibody with chromatin overnight --> Bind CDB-washed Protein A* beads with complexes for 2 hours --> Wash with Low Salt Immune Complex Buffer --> Wash with High Salr Wash Buffer --> Wash with LiCl Immune Complex Wash Buffer --> Wash with TE buffer --> Elute for Western Note: *H3K27me3 ab 07-449 was Protein A purified (Millipore) --> Wash with complete sonication buffer (same as IP using myc-conjugated beads) --> Elute with Western loading buffer

Today: > Prepare complete sonication buffer (for dilution) > Antibody binding --> Use H3K27me3 07-449 --> Use double x-linked KAH126-1 (surplus chromatin, not important)

--> Rotate at 4°C/ overnight


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