Anti-IKK beta antibody [Y466]

• IP

Unknown

Immunoprecipitation - Anti-IKK beta antibody [Y466] (AB32135)

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.

All lanes:

Immunoprecipitation - Anti-IKK beta antibody [Y466] (ab32135)

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

Observed band size: 76 kDa

false

• WB

Lab

Western blot - Anti-IKK beta antibody [Y466] (AB32135)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-IKK beta antibody [Y466] (ab32135) at 1/2500 dilution

All lanes:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 87 kDa

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• WB

Lab

Western blot - Anti-IKK beta antibody [Y466] (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 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.

All lanes:

Western blot - Anti-IKK beta antibody [Y466] (ab32135) 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: 87 kDa

false

• WB

Lab

Western blot - Anti-IKK beta antibody [Y466] (AB32135)

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.

All lanes:

Western blot - Anti-IKK beta antibody [Y466] (ab32135)

Predicted band size: 87 kDa

false

• WB

Lab

Western blot - Anti-IKK beta antibody [Y466] (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] (ab32135) 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

false

• WB

Lab

Western blot - Anti-IKK beta antibody [Y466] (AB32135)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-IKK beta antibody [Y466] (ab32135) at 1/1000 dilution

Lane 1:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 20 µg

Lane 2:

Mouse lung lysates at 20 µg

Lane 3:

Rat brain lysates at 20 µg

Lane 4:

SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 87 kDa

Observed band size: 87 kDa

false

• WB

Unknown

Western blot - Anti-IKK beta antibody [Y466] (AB32135)

unpurified ab32135

All lanes:

Western blot - Anti-IKK beta antibody [Y466] (ab32135)

Predicted band size: 87 kDa

false

Exposure time: 3min

• WB

Unknown

Western blot - Anti-IKK beta antibody [Y466] (AB32135)

All lanes:

Western blot - Anti-IKK beta antibody [Y466] (ab32135) at 1/5000 dilution

All lanes:

Daudi cell lysate

Predicted band size: 87 kDa

Observed band size: 87 kDa

false