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

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05/18/10

  • ✓ 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):

  • 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

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

Gel loading

- PageRuler Plus pre-stained ladder
1. U2OS, 29 μg
2. U2OS, 14 μg
3. U2OS, 7 μg
4. U2OS, 3.5 μg

5/18/10 Ponceau S stain
Ponceau S stained filters


> 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)

Lanes
  1. PageRuler Plus pre-stained ladder
  2. U2OS, 29 mg
  3. U2OS, 14 mg
  4. U2OS, 7 mg
  5. U2OS, 3.5 mg

Signals

  • Blot 1, H3 = 15 kD
  • Blot 2, H3K27me3 = > 15 kD
  • Blot 3, PolII = 217 kD
  • Blot 4, BMI1 = 42 kD
Image:KAH 051910 WB.tif
## min. exposure

> Coclusions:



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