Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: IKK beta knockout HAP1 cell lysate (20 µg)
Lanes 1 and 2: Merged signal (red and green). Green - ab124957 observed at 90 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab124957 was shown to specifically react with IKK beta when IKK beta knockout samples were used. Wild-type and IKK beta knockout samples were subjected to SDS-PAGE. ab124957 and ab8245 (loading control to GAPDH) were diluted 1/500 and 1/2000 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 1/10,000 dilution for 1 h at room temperature before imaging.

ab124957, at 1/100 dilution staining IKK beta in paraffin-embedded Mouse kidney tissue, by Immunohistochemistry.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

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 [EPR6043] (ab124957) at 1/1000 dilution

Lane 1 : HeLa cell lysates
Lane 2 : 293T cell lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled Goat anti-Rabbit IgG at 1/2000 dilution

Predicted band size: 87 kDa

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling IKK beta with unpurified ab124957 at 1/20 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

ab124957, at 1/100 dilution staining IKK beta in paraffin-embedded Human cervix carcinoma tissue, by Immunohistochemistry.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

ab124957, at 1/50 dilution, staining IKK beta in HeLa cells by Immunofluorescence.

Equilibrium disassociation constant (K_D)
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