User:Karmella Haynes/Notebook/Polycomb project/2010/05/12

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05/12/10

 * &#x2713; Western blot: Test ab's for ChIP (finish on 5/13/10)

Western blot

> SDS-PAGE --> 2x 10-well tris-glycine (4-20%) pre-cast gels --> Samples (25 μL total vol./ well): --> 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: 20 μL
 * KAH128-8.2-4, FTRx: 12.5 μL protein

> Electroblot --> Run @ 100 V for 1 hr. --> Cut each filter in half & trim (4 sets of samples total))

> Immunostaining --> Primaries (4°C/ overnight): --> Secondary: donkey anti-rabbit IgG-HRP, 1:5,000 (R.T./ 1 hr.) (Molecular weights computed at http://www.expasy.ch/tools/pi_tool.html)
 * 1) rabbit anti-DsRed (632496), 1:4000, 5 mL
 * 2) rabbit anti-myc (ab9106), 1:5000, 5 mL
 * 3) rabbit anti-H3 (ab1791), 1:5000, 5 mL
 * 4) rabbit anti-H3K27me3 (07-449), 1:5000, 5 mL

> Coclusions: --> KAH128-8.3 shows expression of Pc-ATF. Keep this line. Discard others. --> DsRed antibody: Pc-ATF band might be cross contamination from anti-myc (washed all filters together). Already know this antibody works, so no repeat needed. --> myc antibody: very strong (some background on WB). Use for ChIP. --> H3 antibody: successful blot of histones for 1 hr. electroblot. Good antibody. (Pc-ATF band probably anti-myc contamination) --> H3K27me3 antibody: No bands detected. Probably due to small proportion of methylated histones. Repeat using higher concentration of U2OS protein. (Pc-ATF band probably anti-myc contamination)


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