User:Karmella Haynes/Notebook/Polycomb project/2010/02/05

{| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Project name
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

02/05/10

 * &#x2713; HEK293 #4 Luc: expand to 74 cm2 flask (non selective 10% FBS, 1% P/S DMEM)
 * &#x2713; Polycomb-ATF lines: thaw cells
 * &#x2713; Western: Gal4-EED set (same as 2/03/20); re-probe stripped filter w/ anti-EED; new blot for anti-Gal4

Thaw Polycomb-ATF lines --> Pellet thawed cells in 4 ml 100% FBS --> Plate cells in 20% FBS, 1% P/S McCoy's 5A
 * 1) KAH126-1 (human Pc-CD)
 * 2) KAH127-4 (mouse Pc-CD)
 * 3) KAH128-8 (fly Pc-CD)
 * 4) KAH129-4 (fish Pc-CD)
 * 5) KAH130-2 (human CBX8)
 * 6) KAH131-9 (fly Pc)
 * 7) KAH132-8 mCh-VP64
 * 8) KAH133-1 mCh

Western blot

> Stripped blot #2 (from 2/04) --> Incubate in blocking solution @ R.T./ > 1 hr.

> SDS-PAGE --> Make new blot of Gal4 staining --> 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 --> Run @ 100 V for 1 hr., 15 min.

> Immunostaining, Blot 2 --> Primary: mouse anti-EED, 1:1000, 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)

> Immunostaining, Blot 3 --> Primary: mouse anti-Gal4, 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)


 * }