User:Karmella Haynes/Notebook/Polycomb project/2010/06/11

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06/11/10

  • ✓ ChIP: bead binding, bead wash, elution, quality control Western
  • ✓ Cell culture: split 126-1, 126-3, 132-8, 154-2, FTRx 1:10
  • ✓ ChIP cell cultures: 5x 25 ml plates for 126-1, 154-2, 132-8, FTRx (+ 1μg/mL dox)
  • ✓ RT-PCR: start 6-well plates w/ 126-1, 126-4, 132-8, 154-2, FTRx -/+ 1μg/mL dox; seeded ~250k cells
  • ✓ Plate for Pc-ATF Western: 126-1, 132-8, 154-2 -/+ 1μg/mL dox; seeded ~250k cells

Remember to refresh dox every two days



ChIP quality control Western
> Pronase treatment overnight (continue tomorrow)
> Western blot samples:

  1. 126-1 mouse α-PolII ChIP Supernatant
  2. 126-1 mouse α-PolII ChIP Eluate
  3. 126-1 rabbit α-myc ChIP Supernatant
  4. 126-1 rabbit α-myc ChIP Eluate
  5. 126-1 rabbit α-H3K27me3 ChIP Supernatant
  6. 126-1 rabbit α-H3K27me3 ChIP Eluate
  7. 126-1 mouse/rabbit α-IgG ChIP Supernatant
  8. 126-1 mouse/rabbit α-IgG ChIP Eluate
  9. 126-1 Input
  10. U2OS mouse α-PolII ChIP Supernatant
  11. U2OS mouse α-PolII ChIP Eluate
  12. U2OS rabbit α-myc ChIP Supernatant
  13. U2OS rabbit α-myc ChIP Eluate
  14. U2OS rabbit α-H3K27me3 ChIP Supernatant
  15. U2OS rabbit α-H3K27me3 ChIP Eluate
  16. U2OS mouse/rabbit α-IgG ChIP Supernatant
  17. U2OS mouse/rabbit α-IgG ChIP Eluate
  18. U2OS Input

> Use 10-well gel (loading volume = 25 μL)
> Electroblot: 1 hr. 30 min.

Gel #1
  1. PageRuler Plus pre-stained ladder (10 μL)
  2. 126-1 mouse α-PolII Sup (15 μL)
  3. 126-1 mouse α-PolII Eluate
  4. 126-1 rabbit α-myc Sup
  5. 126-1 rabbit α-myc Eluate
  6. 126-1 rabbit α-H3K27me3 Sup
  7. 126-1 rabbit α-H3K27me3 Eluate
  8. 126-1 mouse/rabbit α-IgG Sup
  9. 126-1 mouse/rabbit α-IgG Eluate
  10. 126-1 Input
Gel #2
  1. PageRuler Plus pre-stained ladder (10 μL)
  2. U2OS mouse α-PolII Sup (15 μL)
  3. U2OS mouse α-PolII Eluate
  4. U2OS rabbit α-myc Sup
  5. U2OS rabbit α-myc Eluate
  6. U2OS rabbit α-H3K27me3 Sup
  7. U2OS rabbit α-H3K27me3 Eluate
  8. U2OS mouse/rabbit α-IgG Sup
  9. U2OS mouse/rabbit α-IgG Eluate
  10. U2OS Input
6/13/10 Ponceau S stain
Ponceau S stained filter

Note: Not very much protein. Grow new 25 mL plates for fresh chromatin preps.


--> Cut filters into strips, group by antibody
--> Block overnight (or longer)


6/13/10
--> Primaries:
1,5. mouse α-PolII 8WG16 ab817, 1:500, 2 mL
2,6. rabbit α-myc ab9106, 1:1000, 2 mL
3,7. rabbit α-H3K27me3 07-449, 1:500, 2 mL
4,8. mouse/rabbit α-IgG sc2025/sc2027 (hold, wait for results from other blots)


6/14/10
--> Secondaries (R.T./ 1 hr.):
1,5. donkey α-mouse IgG-HRP, 1:5,000
2,6. donkey α-rabbit IgG-HRP, 1:5,000
3,7. donkey α-rabbit IgG-HRP, 1:5,000

6/14/10 Western blot

--> Pull-down unsuccessful. Work with Qingqing to optimize procedure.

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