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 - ab183041 observed at 40 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab183041 was shown to specifically react with mH2A1 when mH2A1 knockout samples were used. Wild-type and mH2A1 knockout samples were subjected to SDS-PAGE. Ab183041 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 10000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

 

 

ab183041 staining mH2A1 in HAP1 WT and H2AFY knockout cells. The cells were fixed with 100% methanol (5min), permeabilized with 0.1%PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab183041 at 1μg/ml and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin - Microtubule Marker (Alexa Fluor® 594) at 1/250 dilution (shown in pseudocolor red). Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488) at 1/1000 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling mH2A1 with ab183041 at 1/100 dilution followed by prediluted HRP Polymer for Rabbit IgG. Counter stained with Hematoxylin.

All lanes : Anti-mH2A1 antibody [EPR9359(2)] (ab183041) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : H2AFY knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 40 kDa
Observed band size: 40 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab183041 observed at 40 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab183041 was shown to react with mH2A1 in wild-type HEK-293T cells in western blot. The band observed in knockout cell line ab266241 (knockout cell lysate ab257463) lane below 40kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HEK-293T and H2AFY knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab183041 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-mH2A1 antibody [EPR9359(2)] (ab183041) at 1/50000 dilution

Lane 1 : HepG2 cell lysate
Lane 2 : MCF7 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : 293T cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate at 1/1000 dilution

Predicted band size: 40 kDa
Observed band size: 40 kDa

Immunofluorescent analysis of 4% paraformaldehyde-fixed MCF7 cells labeling mH2A1 with ab183041 at 1/250 dilution followed by Goat anti rabbit IgG (Alexa Fluor® 488) at 1/200 dilution (green). Counter stained with Dapi (blue).

Immunofluorescent analysis of acetone-fixed HeLa cells labeling mH2A1 with ab183041 at 1/250 dilution followed by Goat anti rabbit IgG (Alexa Fluor® 488) at 1/200 dilution (green). Counter stained with Dapi (blue).

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling mH2A1 with ab183041 at 1/100 dilution followed by prediluted HRP Polymer for Rabbit IgG. Counter stained with Hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver from wild-type and mH2A1/2 knock out tissue sections labeling mH2A1 with ab183041 at 1/400 dilution. Sections were fixed in formaldehyde; heat mediated antigen retivial was performed using a citrate buffer pH 6. An undiluted polyclonal horse anti-rabbit IgG (HRP-conjugated) was used as the secondary antibody.

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