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

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08/16/10

 * &#x2713; Senescence assay (replicate #3): flow cytometry
 * &#x2713; Microscopy (IF): fixing/ immunostaining
 * &#x2713; Western: re-do Pc-ATF -/+ dox (15-well gels looked bad, used old gels)
 * &#x2713; 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): --> 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
 * 1) 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

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.

> 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

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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