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.

All lanes : Anti-NFkB p100/NFKB2 antibody [EPR4686] (ab109440) at 1/1000 dilution

Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : NFKB2 knockout HCT116 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 97 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?

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 knockout cell line ab266883 (knockout cell lysate ab257245) lane below 97kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HCT116 and NFKB2 knockout 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.

All lanes : Anti-NFkB p100/NFKB2 antibody [EPR4686] (ab109440) at 1/1000 dilution

Lane 1 : Wild-type HepG2 cell lysate
Lane 2 : NFKB2 knockout HepG2 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 97 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?

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 knockout cell line ab262323 (knockout cell lysate ab257247) lane below 97kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HepG2 and NFKB2 knockout 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.

All lanes : Anti-NFkB p100/NFKB2 antibody [EPR4686] (ab109440) at 1/10000 dilution

Lane 1 : Jurkat cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : ECV-304 cell lysate
Lane 4 : MCF7 cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 97 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?