User:Karmella Haynes/Notebook/Polycomb project/2010/01/21

{| 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"|

01/21/10

 * &#x2713; Western blot: Polycomb-ATF lines, check size of fusion protein w/ anti-myc

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:

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



--> KAH133 (mCherry-only constructs) look good --> Repeat this expt. for all others (seed 12-well plates for new protein preps)


 * }