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

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05/12/10

  • ✓ 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):

  • PageRuler Plus pre-stained ladder: 20 μL
  • KAH128-8.2-4, FTRx: 12.5 μL protein

--> 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


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

Gel loading

- PageRuler Plus pre-stained ladder
1. KAH128-8.2 (4/13/10)
2. KAH128-8.3 (4/13/10)
3. KAH128-8.4 (4/13/10)
4. FTRx (parent line, 2/17/10)

File:KAH 051210 ps.tif
Ponceau S stained filters


> Immunostaining
--> Primaries (4°C/ overnight):

  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

--> 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)

Lanes
  1. PageRuler Plus pre-stained ladder
  2. KAH128-8.2 = 43 kD
  3. KAH128-8.3 = 43 kD
  4. KAH128-8.4 = 43 kD
  5. FTRx (parent line)

Signals

  • Blot 1, DsRed (Pc-ATF fusions) = 43 kD
  • Blot 2, myc (Pc-ATF fusions) = 43 kD
  • Blot 3, H3 (all) = 15 kD
  • Blot 4, H3K27me3 (all) = > 15 kD
File:KAH 051310 WB.tif
15 min. exposure

> 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)