Recombinant Anti-NFkB p100/NFKB2 antibody [EPR4686] (ab109440)

Lane 1: Wild-type HAP1 cell lysate (20 µg)

Lane 2: NFκB p100 knockout HAP1 cell lysate (20 µg)

Lane 3: Jurkat cell lysate (20 µg)

Lane 4: HeLa cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab109440 observed at 100 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab109440 was shown to specifically react with NFκB p100 when NFκB p100 knockout samples were used. Wild-type and NFκB p100 knockout samples were subjected to SDS-PAGE. ab109440 and ab109440 (loading control to GAPDH) were diluted 1/10000 and 1/2000 respectively and incubated overnight at 4°C. 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 1/10000 dilution for 1 h at room temperature before imaging.

ab109440 staining NFkB p100/p52 in wild-type HAP1 cells (top panel) and NFkB p100/p52 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% 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 with ab109440 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Lanes 1- 2: Merged signal (red and green). Green - ab109440 observed at 120 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab109440 was shown to react with NFkB p100/NFKB2 in wild-type HCT116 cells in western blot. The band observed in CRISPR/Cas9 edited cell line ab266883 (CRISPR/Cas9 edited cell lysate ab257245) lane below 97kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HCT116 and NFKB2 CRISPR/Cas9 edited HCT116 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. ab109440 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) 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.

Lanes 1- 2: Merged signal (red and green). Green - ab109440 observed at 120 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab109440 was shown to react with NFkB p100/NFKB2 in wild-type HepG2 cells in western blot. The band observed in CRISPR/Cas9 edited cell line ab262323 (CRISPR/Cas9 edited cell lysate ab257247) lane below 97kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HepG2 and NFKB2 CRISPR/Cas9 edited HepG2 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. ab109440 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) 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.