Anti-IKB alpha antibody [EP697]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IKB alpha antibody [EP697] (AB76429)

Immunohistochemical analysis of Paraffin-embedded human colon tissue section labelling IKB alpha with ab76429 at 1/100 dilution.

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IKB alpha antibody [EP697] (AB76429)

Immunohistochemical analysis of paraffin-embedded human colon tissue sections labelling IKB alpha with purified ab76429 at dilution of 1/500. Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. The sample was counterstained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-IKB alpha antibody [EP697] (AB76429)

Immunocytochemistry/Immunofluorescence staining of HeLa cells labelling IKB alpha with purified ab76429 at a working dilution of 1/250. The secondary antibody was Alexa Fluor® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. DAPI was used as nuclear counterstain. The cells were fixed in 4% Paraformaldehyde and permeabilized using 0.1% Triton X-100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, rabbit primary antibody was used followed by an Alexa Fluor® 594 goat anti-mouse antibody (ab150120). For negative control 2, ab7291 (mouse anti-tubulin) was used followed by an Alexa Fluor® 488 goat anti-rabbit antibody (ab150077).

• WB

Lab

Western blot - Anti-IKB alpha antibody [EP697] (AB76429)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : IKB alpha knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)

Lanes 1 - 3 : Merged signal (red and green). Green - ab76429 observed at 39 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab76429 was shown to specifically react with IKB alpha in wild-type HAP1 cells as well as additional cross-reactive bands. No bands were observed when IKB alpha knockout cells were examined. Wild-type and IKB alpha knockout samples were subjected to SDS-PAGE. ab76429 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/500 dilution and 1/10,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-IKB alpha antibody [EP697] (ab76429)

Predicted band size: 35 kDa

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• WB

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Western blot - Anti-IKB alpha antibody [EP697] (AB76429)

All lanes:

Western blot - Anti-IKB alpha antibody [EP697] (ab76429) at 1/1000 dilution

Lane 1:

HeLa cell lysates at 10 µg

Lane 2:

Jurkat cell lysates at 10 µg

Secondary

All lanes:

goat anti-rabbit HRP at 1/1000 dilution

Predicted band size: 35 kDa

Observed band size: 36 kDa

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• WB

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Western blot - Anti-IKB alpha antibody [EP697] (AB76429)

Blocking/Diluting buffer 5% NFDM /TBST

All lanes:

Western blot - Anti-IKB alpha antibody [EP697] (ab76429) at 1/1000 dilution

Lane 1:

Jurkat (human acute T cell leukemia) whole cell lysate at 15 µg

Lane 2:

HeLa (human cervix adenocarcinoma) whole cell lysate at 15 µg

Secondary

All lanes:

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

Predicted band size: 35 kDa

Observed band size: 39 kDa

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• WB

Unknown

Western blot - Anti-IKB alpha antibody [EP697] (AB76429)

Blocking/Diluting buffer 5% NFDM /TBST

All lanes:

Western blot - Anti-IKB alpha antibody [EP697] (ab76429) at 1/10000 dilution

Lane 1:

RAW264.7 (mouse abelson murine leukemia virus-induced tumor) whole cell lysate at 15 µg

Lane 2:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 35 kDa

Observed band size: 39 kDa

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• WB

CiteAb

Western blot - Anti-IKB alpha antibody [EP697] (AB76429)

IKB alpha western blot using anti-IKB alpha antibody [EP697] ab76429. Publication image and figure legend from Wang, T., Hao, Z., et al., 2020, Cell Death Dis, PubMed 32732957.

ab76429 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab76429 please see the product overview.

Altered MYD88 promotes the activation of NF-κB signaling pathway.a, b The effect of MYD88 on NF-κB pathway was detected by western blot and immunofluorescence staining. c, d CircRNF121 and miR-665 impacted the IRAK-4-binding MYD88 expression. e, f The effect of circRNF121/miR-665/MYD88 axis on NF-κB signaling pathway was detected by western blot. Data were means ± SD of three independent assays (*p < 0.05).

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