User:Karmella Haynes/Notebook/Polycomb project/2010/05/18

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


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05/18/10

 * &#x2713; Western blot: optimize ab's for ChIP

Western blot

> SDS-PAGE --> 2x 10-well tris-glycine (4-20%) pre-cast gels --> Samples (25 μL total vol./ well, dilute protein w/ RIPA): --> Make 100 mM DTT in 2x loading buffer (> enough for 12.5 μL/ gel lane) --> Add 12.5 μL DTT + buffer to each protein sample --> Incubate at 100°C/ 5 min. --> Run gel @ 120 V until dye front reaches the bottom
 * PageRuler Plus pre-stained ladder: 15 μL
 * U2OS, 12.5 μL, ~ 29 μg protein
 * U2OS, 6.0 μL, ~ 14 μg
 * U2OS, 3.0 μL, ~ 7 μg
 * U2OS, 1.5 μL, ~ 3.5 μg

> Electroblot --> Run @ 100 V for 1 hr. (blot 1, histones), and 1.5 hr (blot 2, large proteins) --> Cut each filter in half & trim (4 sets of samples total))

> Immunostaining --> Block 4°C/ overnight; continue Wed. through Fri.

5/19/10 --> Primaries (4°C/ overnight):
 * 1) rabbit anti-H3 (ab1791), 1:5000, 5 mL
 * 2) rabbit anti-H3K27me3 (07-449), 1:1000, 5 mL
 * 3) mouse anti-PolII (ab817), 1:1000, 5 mL
 * 4) mouse anti-BMI1 (ab14389), 1:1000, 5 mL

5/20/10 --> Secondary: 1,2. donkey anti-rabbit IgG-HRP, 1:5,000 (R.T./ 1 hr.) 3,4. donkey anti-mouse IgG-HRP, 1:5,000 (R.T./ 1 hr.) (Molecular weights computed at http://www.expasy.ch/tools/pi_tool.html)

> Coclusions:


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