User:Karmella Haynes/Notebook/Polycomb project/2010/01/27

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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01/27/10

 * &#x2713; Protein preps: dox-induced Pc-ATF lines from 12-well plate
 * &#x2713; Western blot: repeat from 1/25 using max. vol. protein

> 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., 10 min. (for large protein transfer)

> Immunostaining --> Primary: mouse anti-myc, 1:2500, 5 mL (4°C/ overnight) --> Secondary: donkey anti-mouse-HRP, 1:10,000 (R.T./ 1 hr.)


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