User:Karmella Haynes/Notebook/Polycomb project/2010/06/16

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06/16/10

  • ✓ Western: Check histone methylation antibodies; check p16 antibody
  • ✓ Order oligos: For confirming proper Pc-ATF transgene transcripts
  • ✓ Pc-ATF + H3me sensor lines: Pick colonies; transfer to 24-well plate



Task 1

  1. CMVTetO-r2-rc: 5'-ACCGATCCAGCCTCCG (132 bp downstream of TATA box, Use with mCh r1)
  2. Myc r1: 5'-cagatcctcttctgagatgag (21 bp in myc epitope tag, KAH011, Use with mCh f1)



Western blot
> Gel 1: Check H3, H3K27me3, and H3K4me3 antibodies
--> Samples loaded:
1,3,5,7. U2OS plain (34.5 μg, 15 μL)
2,4,6,8. U2OS plain (17.25 μg, 7.5 μL)

> Gel 2: Check p16 antibody (new)
--> Samples loaded:
1. 126-1 (15 μL)
2. 126-1 + dox 4 days (15 μL)
3. 154-2 (15 μL)
4. 154-2 + dox 4 days (15 μL)

> Use 4x loading dye w/ BME (from Qingqing & Mike)
> Use 10-well gel (loading volume = 25 μL)
> Electroblot: 1 hr. 30 min.

Gel #1
  1. PageRuler Plus (10 μL)
  2. U2OS plain (34.5 μg)
  3. U2OS plain (17.25 μg)
  4. U2OS plain (34.5 μg)
  5. U2OS plain (17.25 μg)
  6. U2OS plain (34.5 μg)
  7. U2OS plain (17.25 μg)
  8. U2OS plain (34.5 μg)
  9. U2OS plain (17.25 μg)
  10. PageRuler Plus (10 μL)
Gel #2
  1. PageRuler Plus (10 μL)
  2. 126-1 (15 μL)
  3. 126-1 + dox 4 days (15 μL)
  4. 154-2 (15 μL)
  5. 154-2 + dox (15 μL)
  6. 126-1 (15 μL)
  7. 126-1 + dox 4 days (15 μL)
  8. 154-2 (15 μL)
  9. 154-2 + dox (15 μL)
  10. PageRuler Plus (10 μL)
File:KAH 061610 ps1.tif
Ponceau S stained filters


--> Gel 1: Cut filters into 4 strips (2 samples each)
--> Gel 2: Cut in half (4 samples each)
--> Block at R.T./ 1 hour


6/16/10
--> Primaries:
1.1. rabbit α-H3 ab1791, 1:500, 3 mL
1.2. rabbit α-H3K27me3 07-449, 1:500, 3 mL
1.3. mouse α-H3K27me3 ab6002, 1:500, 3 mL
1.4. rabbit α-H3K4 C42D8, 1:500, 3 mL
2.1. mouse α-p16 ab16880, 1:100, 3 mL
2.2. rabbit α-myc ab9106, 1:5000, 3 mL


6/17/10
--> Secondaries (R.T./ 1 hr.):
1.3, 2.1. donkey α-mouse IgG-HRP, 1:5000, 5 mL
1.1, 1.2, 1.4, 2.2. donkey α-rabbit IgG-HRP, 1:5000, 5 mL

6/17/10 Western blot

> Conclusions

  • rabbit α-H3K27me3 07-449 shows non-specific signal.
  • mouse α-H3K27me3 ab6002 failed (also showed no signal in IF, 4/05/10)
  • rabbit α-H3K4 C42D8 failed.
  • mouse α-p16 ab16880 failed.
  • Wash filters overnight in 1x PBST and repeat staining with gentler blocking buffer (0.5% BSA/PBST).