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

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  • ✓ HEK Gal4EED + Pc-ATF transf.: RFP expression is low, refresh medium and check again tomorrow

--> 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

  • ✓ 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

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

Reagent 1 2
α-H3K27me3 07-449 1.0 ---
rabbit IgG ab --- 1.0
KAH126-1 dx 250.0 250.0
Sonication buffer 250.0 250.0

--> Rotate at 4°C/ overnight