User:Karmella Haynes/Notebook/Polycomb project/2010/02/03

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02/03/10

 * &#x2713; Protein prep: Gal4-EED transfections (from 2/02)
 * &#x2713; Western blot: 1. Polycomb-ATF lines, 2. Gal4-EED transfections
 * &#x2713; HEK293 #4 Luc cells: seeded culture from 2/20 not growing well; change to 20% FBS DMEM; thaw new tube, pellet in FBS, plate in 20% FBS DMEM

Western blot

> SDS-PAGE --> Use 6μL protein for all samples --> Make 100 mM DTT in 2x loading buffer (200 μL total; > enough for 6 μL/ gel lane) --> Add 6 μL DTT + buffer to each protein sample --> Incubate at 100°C/ 5 min. --> Run gel @ 120 V

> Electroblot 1 --> Run @ 100 V for 1 hr., 10 min. (for large protein transfer)

> Electroblot 2 --> Run @ 100 V for 1 hr., 15 min. (EED shows up at 80kD, according to Abcam)

> Immunostaining 1 --> Primary: mouse anti-myc, 1:5000, 5 mL (4°C/ overnight) --> Secondary: donkey anti-mouse-HRP, 1:10,000 (R.T./ 1 hr.) (Molecular weights computed at http://www.expasy.ch/tools/pi_tool.html)

--> KAH130-2, KAH130-7, KAH131-6 are better, cleaner preps using more cells, but band undetectable. Dox induction showed very few RFP+ cells for 130-2 and none for 130-7 and 131-6 --> Repeat Western blot analysis after thawing and expanding select lines

> Immunostaining 2 --> Primary: mouse anti-EED, 1:5000, 5 mL (4°C/ overnight) --> Secondary: (Molecular weights computed at http://www.expasy.ch/tools/pi_tool.html)
 * donkey anti-mouse-HRP, 1:10,000
 * anti-GAPDH-HRP, 1:10,000 (R.T./ 1 hr.)

--> No bands detected. Strip filter w/ 1x PBST (R.T./ overnight) and re-probe with higher conc. anti-EED (tomorrow)


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