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

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06/16/10

 * &#x2713; Western: Check histone methylation antibodies; check p16 antibody
 * &#x2713; Order oligos: For confirming proper Pc-ATF transgene transcripts
 * &#x2713; 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: 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



> 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).


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