Lane 1 : Anti-NFkB p100/NFKB2 antibody [EPR4686-66] (ab175192) at 1/1000 dilution
Lane 2 : Anti-TRIM24 antibody [EPR22825-2] (ab256491) 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 - ab175192 observed at 120 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab175192 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 HCT117 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. ab175192 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 10000 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 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.

Homozygous: 5 bp deletion in exon8

Representative images NFKB2 knockout HCT116 cells, low and high confluency examples (top left and right respectively) and wild-type HCT116 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 M5000 microscope.