Anti-IKK beta antibody [EPR6043]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IKK beta antibody [EPR6043] (AB124957)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IKK beta antibody [EPR6043] (AB124957)

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.

• WB

Lab

Western blot - Anti-IKK beta antibody [EPR6043] (AB124957)

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:

Western blot - Anti-IKK beta antibody [EPR6043] (ab124957) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

IKBKB knockout HeLa cell lysate at 20 µg

Predicted band size: 87 kDa

Observed band size: 90 kDa

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• WB

Lab

Western blot - Anti-IKK beta antibody [EPR6043] (AB124957)

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.

All lanes:

Western blot - Anti-IKK beta antibody [EPR6043] (ab124957) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

IKBKB CRISPR/Cas9 edited HeLa cell lysate at 20 µg

Predicted band size: 87 kDa

Observed band size: 90 kDa

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• WB

Lab

Western blot - Anti-IKK beta antibody [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.

All lanes:

Western blot - Anti-IKK beta antibody [EPR6043] (ab124957) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

IKKa/b knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human IKBKB (IKK beta) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-ikbkb-ikk-beta-knockout-hela-cell-line-ab264847'>ab264847</a>) at 20 µg

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• WB

Lab

Western blot - Anti-IKK beta antibody [EPR6043] (AB124957)

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.

All lanes:

Western blot - Anti-IKK beta antibody [EPR6043] (ab124957)

Predicted band size: 51 kDa,87 kDa

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• WB

Unknown

Western blot - Anti-IKK beta antibody [EPR6043] (AB124957)

All lanes:

Western blot - Anti-IKK beta antibody [EPR6043] (ab124957) at 1/1000 dilution

Lane 1:

HeLa cell lysates at 10 µg

Lane 2:

293T cell lysates at 10 µg

Secondary

All lanes:

HRP labelled Goat anti-Rabbit IgG at 1/2000 dilution

Predicted band size: 87 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-IKK beta antibody [EPR6043] (AB124957)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.