User:Karmella Haynes/Notebook/Polycomb project/2010/07/09

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07/09/10

  • ✓ Western: Pc-ATF lines -/+ dox (for pub figure)
  • ✓ ChIP: myc-beads vs. mouse IgG-beads (132-8 sample)
  • ✓ H3me sensor lines: transfer hygro-resistant colonies to 24-well plates
  • ✓ Cell culture: thaw KAH157-1, 156-5, 158-4, 159-2 (for microscopy & ChIP)



Western
> Western blot loading
> Samples: 15.0 protein + 5.0 4x loading dye
--> Use 4x loading dye (Invitrogen) w/ BME (400 μL loading dye + 40 μL BME)
> Use 15-well gel (loading volume = 15 μL)
> Electroblot: 1 hr. 15 min.

Gel

1. PageRuler Plus (10 μL)
2,3. KAH126-1 -/+ dox (6/15/10)
4,5. KAH154-2 -/+ dox (5/21/10)
6,7. KAH128-8.3 -/+ dox (6/30/10)
8,9. KAH157-1 -/+ dox (5/21/10)
10,11. KAH129-4 -/+ dox (7/02/10)
12,13. KAH156-5 -/+ dox (5/21/10)
14,15. KAH132-8 -/+ dox (6/15/10)

File:KAH 070910 ps1.tif
Ponceau S stained filter

> Block: 5% milk/PBST, R.T./1 hr.
> Primary staining: 5% milk/PBST, 4°C/o.n.
--> rabbit α-myc ab9106, 1:1000, 5 mL


7/01/10
> Secondary staining: 5% milk/PBST, R.T./ 1 hr.
--> donkey α-rabbit, 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
  • KAH129-4 = 43 kD
  • KAH156-5 = 37.5 kD
  • KAH132-8 = 35 kD

File:KAH 071010 WB.tif
20 second exposure, BioRad imager (Chemiluminescence, camera)

--> Conclusion: dox induction looks great for all samples. Wash in 1xPBST o/n for GAPDH-HRP staining tomorrow. Do another gel to fix smeary transfer problem in last three lanes.


7/11/10 File:KAH 071110 WB.tif
αGAPDH-HRP, 1:10,000, 5% milk/PBST; 20 second exposure, BioRad imager (Chemiluminescence, camera)

--> Uneven signal; Try 1:5000 anitbody next time. GAPDH band is shorter than shortest full-length Pc-ATF band; add αGAPDH-HRP to secondary hyb. next time.



ChIP: myc-bead pull-down
> See 6/29/10
--> Use 132-8 sonicated ~6 mL sample prepped according to Qingqing's protocol (+ PLAAC, PMSF, detergent, salt, etc.)
--> Binding:

  1. 500 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
  2. 500 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry

--> Rotate at 4°C overnight
--> Wash and do Western tomorrow



H3me sensor lines
> About 20 colonies on the transfection plates, none on the negative control
> Pick 12 colonies from each plate
> Lines:

  1. KAH96
  2. KAH142
  3. KAH146
  4. KAH147

> Non-clonal back-up stocks (6-well plates) and neg. control: change medium to hygro selection (100 μg/mL hygromycin)
--> Will use these for Gal4-VP64 transfection to do a quick check for functional H3me reporter