Anti-Histone H2A.Z 抗体 - ChIP Grade (ab4174)

Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The  ChIP was performed with 25 µg of chromatin, 2 µg of  ab4174 (blue), and 20 µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

The staining has been done on mouse embryonic cells. The fixation is 2% PFA, and permeabilization is PBS 0.5% triton BSA. The dilution used was 1 in 100 (but it could be used at 1/200 to1/300)

Red  = H2A.Z
Blue  = toto3 for the DNA

Lane 1 Primary: ab4174, 1 ug/ml
Sample: Calf Thymus Histone Preparation Nuclear Lysate, 0.5 ug
Secondary: ab65484, 1/3000 dilution

Lane 2 Primary: ab4174, 1 ug/ml
Sample: HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate, 10 ug
Secondary: ab65484, 1/3000 dilution

Lane 3 Primary: ab4174, 1 ug/ml
Sample: NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate, 10 ug
Secondary: ab65484, 1/3000 dilution

Lane 4 Primary: ab4174, 1 ug/ml
Sample: PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate , 10 ug
Secondary: ab65484, 1/3000 dilution

ICC/IF image of ab4174 stained HepG2 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab4174, 5µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Blocked with 3% BSA for 1 hour at 20°C.

Primary incubation in TBS tween + 3% BSA at 20°C for 1 hour.

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