All lanes : Anti-IKK beta antibody [EPR6043] (ab124957) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : IKBKB knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 87 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab124957 observed at 90 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab124957 was shown to react with IKK beta in wild-type HeLa cells in western blot. The band observed in knockout cell line ab264847 (knockout cell lysate ab257228) lane below 90kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and IKBKB knockout HeLa 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. ab124957 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 500 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-IKK beta antibody [Y466] (ab32135) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : IKBKB knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 87 kDa
Observed band size: 87 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab32135 observed at 87 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab32135 was shown to react with IKK beta in wild-type HeLa cells in western blot. The band observed in knockout cell line ab264847 (knockout cell lysate ab257228) lane below 87kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and IKBKB knockout HeLa 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. ab32135 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: 14 bp deletion in exon 6.