All lanes : Anti-AMF antibody [1B7D7] (ab66340) at 1/1000 dilution

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

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 63 kDa
Observed band size: 63 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab66340 observed at 63 kDa. Red - loading control ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) observed at 37 kDa.

ab66340 was shown to react with AMF in wild-type HEK-293T cells in Western blot with loss of signal observed in GPI knockout cell line ab266834 (GPI knockout cell lysate ab257458). Wild-type HEK-293T and GPI knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab66340 and ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

All lanes : Anti-AMF antibody [EPR11663(B)] (ab167394) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : GPI knockout HEK293T cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 63 kDa
Observed band size: 63 kDa

Lanes 1-4: Merged signal (red and green). Green - ab167394 observed at 63 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab167394 Anti-AMF antibody [EPR11663(B)] was shown to specifically react with AMF in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266834 (knockout cell lysate ab257458) was used. Wild-type and AMF knockout samples were subjected to SDS-PAGE. ab167394 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2.5 hours at 1 in 1000 dilution and 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: 76 bp deletion in exon2