Anti-IKK beta antibody [Y466] - BSA and Azide free

• IP

Unknown

Immunoprecipitation - Anti-IKK beta antibody [Y466] - BSA and Azide free (AB171363)

ab32135 (purified) at 1 : 50 dilution (2μg) immunoprecipitating IKK beta in SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate.

Lane 1 (input) : SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate 10ug
Lane 2 (+) : ab32135 & SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32135 in SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1 : 10,000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32135).

All lanes:

Immunoprecipitation - Anti-IKK beta antibody [Y466] (<a href='/en-us/products/primary-antibodies/ikk-beta-antibody-y466-ab32135'>ab32135</a>)

Predicted band size: 75 kDa,76 kDa,87 kDa

Observed band size: 76 kDa

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• WB

Lab

Western blot - Anti-IKK beta antibody [Y466] - BSA and Azide free (AB171363)

This data was developed using the same antibody clone in a different buffer formulation (ab32135).

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 CRISPR/Cas9 edited cell line ab264847 (CRISPR/Cas9 edited cell lysate ab257228) lane below 87kDa 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. 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.

All lanes:

Western blot - Anti-IKK beta antibody [Y466] (<a href='/en-us/products/primary-antibodies/ikk-beta-antibody-y466-ab32135'>ab32135</a>) 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: 87 kDa

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• WB

Supplier Data

Western blot - Anti-IKK beta antibody [Y466] - BSA and Azide free (AB171363)

Lanes 1, 3 and 5 : Wild-type HAP1 cell lysate (20 μg)
Lanes 2, 4 and 6 : IKK beta knockout HAP1 cell lysate (20 μg)
Lanes 1 and 2 : Green signal from target – ab32135 observed at 87 kDa
Lanes 3 and 4 : Red signal from loading control – ab8226 observed at 42 kDa
Lanes 5 and 6 : Merged (red and green) signal

Unpurified ab32135 was shown to react with IKK beta when IKK beta knockout samples were used, along with additional cross-reactive bands. Wild-type and IKK beta knockout samples were subjected to SDS-PAGE. ab32135 and ab8226 (loading control to beta actin) were diluted 1/1000 and 1/2000 respectively 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32135).

All lanes:

Western blot - Anti-IKK beta antibody [Y466] - BSA and Azide free (ab171363)

Predicted band size: 87 kDa

false