Anti-IKK alpha antibody [Y463]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-IKK alpha antibody [Y463] (AB32041)

Intracellular Flow Cytometry analysis of Daudi (Human Burkitt's lymphoma lymphoblast) cells labelling IKK alpha with purified ab32041 at 1/20 dilution (10 μg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-IKK alpha antibody [Y463] (AB32041)

Overlay histogram showing HAP1 wildtype (green line) and HAP1-CHUK knockout cells (red line) stained with ab32041. The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab32041, 1μg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) presorbed (ab150081) at 1/2000 dilution for 30 min at 22°C. A rabbit IgG isotype control antibody (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-CHUK knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. This antibody can also be used in HAP1 cells fixed with 80% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IKK alpha antibody [Y463] (AB32041)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian cancer tissue sections labeling IKK alpha with purified ab32041 at 1/50 dilution (3.92 µg/mL). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.

• IP

Unknown

Immunoprecipitation - Anti-IKK alpha antibody [Y463] (AB32041)

Purified ab32041 at 1/60 dilution (2μg) immunoprecipitating IKK alpha in HeLa whole cell lysate.
Lane 1 (input) : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg
Lane 2 (+) : ab32041 + HeLa whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab169743 in HeLa whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 88 kDa

All lanes:

Immunoprecipitation - Anti-IKK alpha antibody [Y463] (ab32041)

Predicted band size: 84 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IKK alpha antibody [Y463] (AB32041)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue sections labeling IKK alpha with purified ab32041 at 1/50 dilution (3.92 µg/mL). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.

• WB

Lab

Western blot - Anti-IKK alpha antibody [Y463] (AB32041)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : IKK alpha knockout HAP1 cell lysate (20 μg)
Lanes 1 and 2 : Merged signal (red and green). Green - ab32041 observed at 84 kDa. Red - loading control, ab8226, observed at 42 kDa.

ab32041 (Unpurified format) was shown to specifically react with IKK alpha when IKK alpha knockout samples were used. Wild-type and IKK alpha knockout samples were subjected to SDS-PAGE. ab32041 and ab8226 (loading control to beta actin) were diluted 1/10000 and 1/1000 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-IKK alpha antibody [Y463] (ab32041)

Predicted band size: 84 kDa

false

• WB

Unknown

Western blot - Anti-IKK alpha antibody [Y463] (AB32041)

All lanes:

Western blot - Anti-IKK alpha antibody [Y463] (ab32041) at 1/10000 dilution

All lanes:

Daudi (Human Burkitt's lymphoma lymphoblast) whole cell lysate

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/20000 dilution

Predicted band size: 84 kDa

false

• WB

Unknown

Western blot - Anti-IKK alpha antibody [Y463] (AB32041)

All lanes:

Western blot - Anti-IKK alpha antibody [Y463] (ab32041) at 1/1000 dilution

Lane 1:

RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 15 µg

Lane 2:

Mouse kidney lysate at 15 µg

Lane 3:

C6 (Rat glial tumor glial cell) whole cell lysate at 15 µg

Lane 4:

Rat kidney 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: 84 kDa

false

• WB

Unknown

Western blot - Anti-IKK alpha antibody [Y463] (AB32041)

All lanes:

Western blot - Anti-IKK alpha antibody [Y463] (ab32041) at 1/10000 dilution

All lanes:

Western blot - Recombinant human IKK alpha protein (Active) (<a href='/en-us/products/proteins-peptides/recombinant-human-ikk-alpha-protein-active-ab102103'>ab102103</a>) at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 84 kDa

true

Exposure time: 10s