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:
- Histone H3 ab1791 (rabbit)
- H3K27me3 07-449 (rabbit)
- H3K9me3 9754 (rabbit)
- H3K4me3 9751 (rabbit)
- Bmi1 ab14389 (mouse)
- 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):
- 1-4: Alexafluor 488 goat α-rabbit, 1:1000; Hoescht 33325, 1:1000
- 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
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)
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Ponceau S stained filters; forgot to put top sponge on transfer #2
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> Block: 5% milk/PBST, R.T./1 hr.
> Primary staining: 5% milk/PBST, 4°C/o.n.
- rabbit α-DsRed 632496, 1:2000, 5 mL
- 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
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.
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