Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: mH2A1 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: HepG2 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab91528 observed at 40 kDa. Red - loading control, ab176560, observed at 50 kDa.

ab91528 was shown to specifically recognize mH2A1 in wild-type HAP1 cells. No band was observed when mH2A1 knockout cells were examined. Wild-type and mH2A1 knockout samples were subjected to SDS-PAGE. ab91528 and ab176560 (Rabbit anti-alpha Tubulin loading control) were incubated overnight at 4°C at 10 µg/ml and 1/10,000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

Overlay histogram showing HeLa cells stained with ab91528 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab91528, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Chromatin was prepared from SK-OV-3 cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab91528 (red), or 5 µg of mouse normal IgG1 ab18443 (gray) and 25 µl of Protein A/G Dyna beads. 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 commercial primers from Paper PMID: 22589551
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

All lanes : Anti-mH2A1 antibody [14G7] (ab91528) at 10 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
Lane 3 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed at 1/3000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 40 kDa
Observed band size: 40 kDa
Additional bands at: 14 kDa, 18 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 2 minutes