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

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07/09/10

 * &#x2713; Western: Pc-ATF lines -/+ dox (for pub figure)
 * &#x2713; ChIP: myc-beads vs. mouse IgG-beads (132-8 sample)
 * &#x2713; H3me sensor lines: transfer hygro-resistant colonies to 24-well plates
 * &#x2713; 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.

> 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

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 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: --> Rotate at 4°C overnight --> Wash and do Western tomorrow
 * 1) 500 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
 * 2) 500 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry

H3me sensor lines > About 20 colonies on the transfection plates, none on the negative control > Pick 12 colonies from each plate > Lines: > 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
 * 1) KAH96
 * 2) KAH142
 * 3) KAH146
 * 4) KAH147


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