User:Karmella Haynes/Notebook/Polycomb project/2010/01/25

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01/25/10

 * &#x2713; Polycomb-ATF lines: frozen stocks for confluent plates KAH
 * &#x2713; Cultures for Western blot: 12-well dish (~2.5x105/well) for cell lines above
 * &#x2713; Western blot: KAH126-1, -3, -4; KAH127-3, -4; KAH128-2, -6, -8; KAH129-3, -4, -8; KAH132-8; KAH133-6

Western blot > Bradford assay --> Calculate amount of protein to load in each lane (6 μL max)



> SDS-PAGE --> Bring protein vol. up to 6μL w/ dH2O --> 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. --> Gel loading:

> Electroblot --> Run @ 100 V for 1 hr., 15 min. (for large protein transfer)

> Immunostaining --> Primary: mouse anti-myc, 1:5000 (4°C/ overnight) --> Secondary: donkey anti-mouse-HRP, 1:10,000 (R.T./ 1 hr.)

Results: Blot showed no signal in any lanes, except for a very faint signal in lane 14. Try again with max. volume of protein.


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