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.

Homozygous: 13 bp deletion in exon 2

Representative images of H2AFY knockout HEK293T cells, low and high confluency examples (top left and right respectively) and wild-type HEK293T cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using a EVOS XL Core microscope.