重组Anti-IKK beta抗体[EPR6043] (ab124957)

Western blot: Anti-IKKa/b antibody [EPR6043] (ab124957) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab124957 was shown to bind specifically to IKKa/b. A band was observed at 85 kDa in wild-type HeLa cell lysates with no signal observed at this size in IKKa/b knockout cell line. To generate this image, wild-type and IKKa/b knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

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.

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 CRISPR/Cas9 edited cell line ab264847 (CRISPR/Cas9 edited cell lysate ab257228) lane below 90kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and IKBKB CRISPR/Cas9 edited 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.

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.