Recombinant Anti-IKK alpha antibody [Y463] (ab32041)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [Y463] to IKK alpha
- Suitable for: Flow Cyt (Intra), WB, IHC-P, IP
- Knockout validated
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-IKK alpha antibody [Y463]
See all IKK alpha primary antibodies -
Description
Rabbit monoclonal [Y463] to IKK alpha -
Host species
Rabbit -
Specificity
The rat recommendation is based on the WB results. We do not guarantee IHC-P for rat.
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Tested applications
Suitable for: Flow Cyt (Intra), WB, IHC-P, IPmore details -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: HAP1-WT; Daudi,RAW 264.7 and C6 whole cell lysate. Mouse and rat kidney lysate. Flow Cyt (intra): HAP1-WT and Daudi cells; IP: HeLa cell lysate; IHC-P: Human ovarian cancer and mouse kidney tissue.
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General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
Y463 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Positive Controls
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Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab32041 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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Flow Cyt (Intra) |
1/20.
For unpurified use at 1/50. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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WB | (4) |
1/1000. Detects a band of approximately 88 kDa (predicted molecular weight: 85 kDa).
For unpurified use at 1/10000 - 1/50000. |
IHC-P |
1/50. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
See IHC antigen retrieval protocols. The rat recommendation is based on the WB results. We do not guarantee IHC-P for rat. |
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IP | (1) |
1/60.
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Notes |
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Flow Cyt (Intra)
1/20. For unpurified use at 1/50. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
WB
1/1000. Detects a band of approximately 88 kDa (predicted molecular weight: 85 kDa). For unpurified use at 1/10000 - 1/50000. |
IHC-P
1/50. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. See IHC antigen retrieval protocols. The rat recommendation is based on the WB results. We do not guarantee IHC-P for rat. |
IP
1/60. |
Target
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Function
Acts as part of the IKK complex in the conventional pathway of NF-kappa-B activation and phosphorylates inhibitors of NF-kappa-B thus leading to the dissociation of the inhibitor/NF-kappa-B complex and ultimately the degradation of the inhibitor. As part of the non-canonical pathway of NF-kappa-B activation, the MAP3K14-activated CHUK/IKKA homodimer phosphorylates NFKB2/p100 associated with RelB, inducing its proteolytic processing to NFKB2/p52 and the formation of NF-kappa-B RelB-p52 complexes. Also phosphorylates NCOA3. Phosphorylates 'Ser-10' of histone H3 at NF-kappa-B-regulated promoters during inflammatory responses triggered by cytokines. -
Tissue specificity
Widely expressed. -
Involvement in disease
Defects in CHUK are the cause of cocoon syndrome (COCOS) [MIM:613630]; also known as fetal encasement syndrome. COCOS is a lethal syndrome characterized by multiple fetal malformations including defective face and seemingly absent limbs, which are bound to the trunk and encased under the skin. -
Sequence similarities
Belongs to the protein kinase superfamily. Ser/Thr protein kinase family. I-kappa-B kinase subfamily.
Contains 1 protein kinase domain. -
Post-translational
modificationsPhosphorylated by MAP3K14/NIK, AKT and to a lesser extent by MEKK1, and dephosphorylated by PP2A. Autophosphorylated.
Acetylation of Thr-179 by Yersinia yopJ prevents phosphorylation and activation, thus blocking the I-kappa-B signaling pathway. -
Cellular localization
Cytoplasm. Nucleus. Shuttles between the cytoplasm and the nucleus. - Information by UniProt
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Database links
- Entrez Gene: 1147 Human
- Entrez Gene: 309361 Rat
- Omim: 600664 Human
- SwissProt: O15111 Human
- SwissProt: Q60680 Mouse
- Unigene: 198998 Human
- Unigene: 3996 Mouse
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Alternative names
- chuk antibody
- CHUK1 antibody
- Conserved Helix Loop Helix Ubiquitous Kinase antibody
see all
Images
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All lanes : Anti-IKK alpha antibody [Y463] (ab32041) at 1/1000 dilution (Purified)
Lane 1 : RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 2 : Mouse kidney lysate
Lane 3 : C6 (Rat glial tumor glial cell) whole cell lysate
Lane 4 : Rat kidney lysate
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 85 kDa -
Anti-IKK alpha antibody [Y463] (ab32041) at 1/10000 dilution + Daudi (Human Burkitt's lymphoma lymphoblast) whole cell lysate
Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/20000 dilution
Predicted band size: 85 kDa -
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.
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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.
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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.
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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).
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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.
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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
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
References (67)
ab32041 has been referenced in 67 publications.
- He T et al. The HDAC inhibitor GCJ-490A suppresses c-Met expression through IKKα and overcomes gefitinib resistance in non-small cell lung cancer. Cancer Biol Med 19:1172-92 (2022). PubMed: 35188360
- Guo Y et al. Tripartite motif 52 (TRIM52) promotes proliferation, migration, and regulation of colon cancer cells associated with the NF-κB signaling pathway. J Gastrointest Oncol 13:1097-1111 (2022). PubMed: 35837156
- Lu Y et al. NF-κB and AP-1 are required for the lipopolysaccharide-induced expression of MCP-1, CXCL1, and Cx43 in cultured rat dorsal spinal cord astrocytes. Front Mol Neurosci 15:859558 (2022). PubMed: 35966011
- Ji W et al. Triptolide attenuates inhibition of ankylosing spondylitis-derived mesenchymal stem cells on the osteoclastogenesis through modulating exosomal transfer of circ-0110634. J Orthop Translat 36:132-144 (2022). PubMed: 36185580
- Zheng J et al. β-Hydroxy-β-Methylbutyric Acid Promotes Repair of Sheep Myoblast Injury by Inhibiting IL-17/NF-κB Signaling. Int J Mol Sci 24:N/A (2022). PubMed: 36613892