User:Karmella Haynes/Notebook/Polycomb project/2010/08/16

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08/16/10

  • ✓ Senescence assay (replicate #3): flow cytometry
  • ✓ Microscopy (IF): fixing/ immunostaining
  • ✓ Western: re-do Pc-ATF -/+ dox (15-well gels looked bad, used old gels)
  • ✓ Freeze: KAH130-8, 130-18, 96-11, 142-5, 146-1



Microscopy (IF)
> KAH158-4 cells (hCBX8-RFP, no VP64)
> Antibodies:

  1. Histone H3 ab1791 (rabbit)
  2. H3K27me3 07-449 (rabbit)
  3. H3K9me3 9754 (rabbit)
  4. H3K4me3 9751 (rabbit)
  5. Bmi1 ab14389 (mouse)
  6. PolII 8WG16 (mouse)

> Fix cells w/ 4% formaldehyde (37% solution)/PBS; permeabilize w/ 0.5% TritonX-100/PBS
> Block w/ 5% normal horse serum
> Primary: Stain KAH158-4 cells with 1:100, 1:200, or 1:400 dilutions of antibody (in 5% normal horse serum)
--> Invert cover slips onto 25 μL diluted ab on parafilm; 4°C/ overnight

8/17/10
> Wash 3x (5 min.) w/ 1x PBS
> Wash 1x w/ 5% normal horse serum
> Secondary (in 5% normal horse serum):

  1. 1-4: Alexafluor 488 goat α-rabbit, 1:1000; Hoescht 33325, 1:1000
  2. 5,6: Alexafluor 488 goat α-mouse, 1:1000; Hoescht 33325, 1:1000

--> Invert cover slips onto 25 μL diluted ab on parafilm; R.T./ 1 hr. > Wash 3x (5 min.) w/ 1x PBS
> Mount onto glass slides using Vinol; store at R.T. in the dark


8/21/10

File:KAH 082110 micro1.tif

Conclusion: Use 1:250 dilution for all antibodies


Western
> Samples: 37.5 protein + 12.5 4x loading dye (enough for 2 wells each)
--> 4x loading dye (Invitrogen) w/ freshly added DTT (100 mM final)
> Use 10-well gel (loading volume = 25 μL)
> Electroblot: 1 hr. 15 min.

Gels 1 & 2

1. PageRuler Plus (10 μL)
2,3. KAH126-1 -/+ dox
4,5. KAH154-2 -/+ dox
6,7. KAH128-8.3 -/+ dox
8,9. KAH157-1 -/+ dox
10. FTRx (-dox)

File:KAH 081610 ps1.tif
Ponceau S stained filters; forgot to put top sponge on transfer #2

> Block: 5% milk/PBST, R.T./1 hr.

> Primary staining: 5% milk/PBST, 4°C/o.n.

  1. rabbit α-DsRed 632496, 1:2000, 5 mL
  2. rabbit α-myc ab9106, 1:5000, 5 mL


8/17/10
> Secondary staining: 5% milk/PBST, R.T./ 1 hr.
--> donkey α-rabbit-HRP, 1:5000; mouse α-GAPDH-HRP, 1:5000, 5 mL
> Predicted sizes (using http://www.expasy.ch/tools/pi_tool.html)

  • KAH126-1 = 43 kD
  • KAH154-2 = 37.5 kD
  • KAH128-8.3 = 43 kD
  • KAH157-1 = 37.5 kD


File:KAH 081710 WB1.tif
6 min. exposure, BioRad imager (Chemiluminescence, camera)

--> Conclusion: Myc staining looks better than DsRed. Strip DsRed filter (#1, R.T./1xPBST/Overnight) and re-probe with myc.