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Cell Biology Apoptosis Intracellular NFkB IKK
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Validated using a knockout cell lineRecombinantRabMAb

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

  • Datasheet
Reviews (5)Q&A (3)References (30)

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Western blot - Anti-IKK alpha antibody [Y463] (ab32041)
  • Flow Cytometry - Anti-IKK alpha antibody [Y463] (ab32041)
  • Immunocytochemistry/ Immunofluorescence - Anti-IKK alpha antibody [Y463] (ab32041)
  • Immunoprecipitation - Anti-IKK alpha antibody [Y463] (ab32041)
  • Western blot - Anti-IKK alpha antibody [Y463] (ab32041)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IKK alpha antibody [Y463] (ab32041)
  • Flow Cytometry - Anti-IKK alpha antibody [Y463] (ab32041)
  • 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: WB, IHC-P, Flow Cyt, IP, ICC/IF
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

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Overview

  • Product name

    Anti-IKK alpha antibody [Y463]
    See all IKK alpha primary antibodies
  • Description

    Rabbit monoclonal [Y463] to IKK alpha
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-P, Flow Cyt, IP, ICC/IFmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human IKK alpha aa 1-100 (N terminal). The exact sequence is proprietary.

  • Positive control

    • WB: Daudi and wild-type HAP1 cell lysates. IHC-P: Human stomach carcinoma. ICC/IF: Wild-type HAP1 cells. Flow Cyt: HeLa cells.HAP1-WT cells. IP: HeLa cell lysate
  • 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
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 49% PBS, 50% Glycerol (glycerin, glycerine), 0.05% BSA
  • Concentration information loading...
  • Clonality

    Monoclonal
  • Clone number

    Y463
  • Isotype

    IgG
  • Research areas

    • Cell Biology
    • Apoptosis
    • Intracellular
    • NFkB
    • IKK
    • Signal Transduction
    • Protein Phosphorylation
    • Ser / Thr Kinases
    • Other Kinases
    • Signal Transduction
    • Signaling Pathway
    • Nuclear Signaling
    • NFkB Pathway
    • Cardiovascular
    • Atherosclerosis
    • Vascular Inflammation
    • Inflammatory mediators
    • Immunology
    • Innate Immunity
    • TLR Signaling
    • Cancer
    • Cell Death
    • Apoptosis
    • Apoptosis Markers
    • NFkB
    • IKK

Associated products

  • Alternative Versions

    • Anti-IKK alpha antibody [Y463] - BSA and Azide free (ab169743)
    • Alexa Fluor® 488 Anti-IKK alpha antibody [Y463] (ab200412)
    • Alexa Fluor® 647 Anti-IKK alpha antibody [Y463] (ab200414)
    • HRP Anti-IKK alpha antibody [Y463] (ab200415)
    • PE Anti-IKK alpha antibody [Y463] (ab210716)
    • APC Anti-IKK alpha antibody [Y463] (ab221910)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
    • Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed (ab96899)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • Positive Controls

    • Daudi whole cell lysate (ab3951)
  • Recombinant Protein

    • Recombinant human IKK alpha protein (Active) (ab102103)

Applications

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
WB 1/10000 - 1/50000. Detects a band of approximately 88 kDa (predicted molecular weight: 85 kDa).
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt 1/50.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

IP 1/60.
ICC/IF 1/500.

Target

  • 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
    modifications

    Phosphorylated 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.
  • Target information above from: UniProt accession O15111 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • 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
    • Alternative names

      • chuk antibody
      • CHUK1 antibody
      • Conserved Helix Loop Helix Ubiquitous Kinase antibody
      • Conserved helix loop ubiquitous kinase antibody
      • Conserved helix-loop-helix ubiquitous kinase antibody
      • I Kappa B Kinase 1 antibody
      • I Kappa B Kinase Alpha antibody
      • I-kappa-B kinase 1 antibody
      • I-kappa-B kinase alpha antibody
      • IkappaB kinase antibody
      • IkB kinase alpha subunit antibody
      • IkBKA antibody
      • IKK 1 antibody
      • IKK A antibody
      • IKK a kinase antibody
      • IKK-A antibody
      • IKK-alpha antibody
      • IKK1 antibody
      • IKKA antibody
      • IKKA_HUMAN antibody
      • Inhibitor Of Kappa Light Polypeptide Gene Enhancer In B Cells antibody
      • Inhibitor Of Nuclear Factor Kappa B Kinase Alpha Subunit antibody
      • Inhibitor of nuclear factor kappa-B kinase subunit alpha antibody
      • NFKBIKA antibody
      • Nuclear Factor Kappa B Inhibitor Kinase Alpha antibody
      • Nuclear factor NF kappa B inhibitor kinase alpha antibody
      • Nuclear factor NF-kappa-B inhibitor kinase alpha antibody
      • Nuclear factor NFkappaB inhibitor kinase alpha antibody
      • Nuclear Factor Of Kappa Light Chain Gene Enhancer In B Cells Inhibitor antibody
      • TCF-16 antibody
      • TCF16 antibody
      • Transcription factor 16 antibody
      see all

    Images

    • Western blot - Anti-IKK alpha antibody [Y463] (ab32041)
      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 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 1/10 000 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 1/10 000 dilution for 1 h at room temperature before imaging.

    • Flow Cytometry - Anti-IKK alpha antibody [Y463] (ab32041)
      Flow Cytometry - 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 with80% methanol (5 min), , permeabilized with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

    • Immunocytochemistry/ Immunofluorescence - Anti-IKK alpha antibody [Y463] (ab32041)
      Immunocytochemistry/ Immunofluorescence - Anti-IKK alpha antibody [Y463] (ab32041)

      ab32041 staining IKK alpha in wild-type HAP1 cells (top panel) and IKK alpha knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab32041 at 1/500 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

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

    • Western blot - Anti-IKK alpha antibody [Y463] (ab32041)
      Western blot - Anti-IKK alpha antibody [Y463] (ab32041)
      Anti-IKK alpha antibody [Y463] (ab32041) at 1/50000 dilution + Daudi cell lysate

      Predicted band size: 85 kDa
      Observed band size: 88 kDa
      why is the actual band size different from the predicted?

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IKK alpha antibody [Y463] (ab32041)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IKK alpha antibody [Y463] (ab32041)

      Ab32041, at a dilution of 1/50, staining IKK alpha in paraffin embedded human stomach carcinoma by Immunohistochemisty.

      Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

    • Flow Cytometry - Anti-IKK alpha antibody [Y463] (ab32041)
      Flow Cytometry - Anti-IKK alpha antibody [Y463] (ab32041)
      Overlay histogram showing HeLa cells stained with ab32041 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32041, 1/50 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

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

    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.

    Click here to view the general protocols

    Datasheets and documents

    • Datasheet
  • References (30)

    Publishing research using ab32041? Please let us know so that we can cite the reference in this datasheet.

    ab32041 has been referenced in 30 publications.

    • Tang H  et al. CDK5 inhibition in vitro and in vivo induces cell death in myeloma and overcomes the obstacle of bortezomib resistance. Int J Mol Med 45:1661-1672 (2020). PubMed: 32236619
    • Cheng R  et al. Periodontal inflammation recruits distant metastatic breast cancer cells by increasing myeloid-derived suppressor cells. Oncogene 39:1543-1556 (2020). PubMed: 31685946
    • Xie B  et al. miR-135a Alleviates Silica-Induced Pulmonary Fibrosis by Targeting NF-?B/Inflammatory Signaling Pathway. Mediators Inflamm 2020:1231243 (2020). PubMed: 32617074
    • Ma Y  et al. 1-Deoxynojirimycin in Mulberry (Morus indica L.) Leaves Ameliorates Stable Angina Pectoris in Patients With Coronary Heart Disease by Improving Antioxidant and Anti-inflammatory Capacities. Front Pharmacol 10:569 (2019). PubMed: 31164826
    • Yang BY  et al. Datura Metel L. Ameliorates Imiquimod-Induced Psoriasis-Like Dermatitis and Inhibits Inflammatory Cytokines Production through TLR7/8-MyD88-NF-?B-NLRP3 Inflammasome Pathway. Molecules 24:N/A (2019). PubMed: 31181689
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    1-8 of 8 Abreviews or Q&A

    Immunoprecipitation abreview for Anti-IKK alpha antibody [Y463]

    Average
    Abreviews
    Abreviews
    abreview image
    Application
    Immunoprecipitation
    Sample
    Mouse Cell lysate - whole cell (Raw 264.7)
    Total protein in input
    100 µg
    Specification
    Raw 264.7
    Treatment
    2 hrs LPS
    Immuno-precipitation step
    Protein G
    Read More

    Abcam user community

    Verified customer

    Submitted Jul 31 2012

    Western blot abreview for Anti-IKK alpha antibody [Y463]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Raw 264.7)
    Loading amount
    50 µg
    Specification
    Raw 264.7
    Treatment
    2 hrs LPS
    Gel Running Conditions
    Reduced Denaturing (10%)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
    Read More

    Abcam user community

    Verified customer

    Submitted Jun 08 2012

    Western blot abreview for Anti-IKK alpha antibody [Y463]

    Average
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Guinea pig Tissue lysate - whole (myometrium pregnant and non-pregnant)
    Loading amount
    30 µg
    Specification
    myometrium pregnant and non-pregnant
    Gel Running Conditions
    Reduced Denaturing (10%)
    Blocking step
    Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 24°C
    Read More

    Abcam user community

    Verified customer

    Submitted Dec 14 2011

    Question

    Do the following antibodies recognize an extracellular epitope or do you have to follow an intracellular protocol for flow? ab238, 80392, 40752, 32041, 76429, 32135, 76311, 10684, 32536, 32360

    Read More

    Abcam community

    Verified customer

    Asked on Jan 12 2011

    Answer

    Thank you for contacting us. Flow cytometry protocols are recommended below. For most of the antibodies we are not able to provide specific epitope information but it seems most targets are intracellular. Hence not on the membrane and cells need fixation prior to staining. ab238- intracellular target. Intracellular protocol is recommended for flow cytometry. ab10684 -intracellular target. Intracellular protocol is recommended for flow cytometry. ab32041- intracellular target. Intracellular protocol is recommended for flow cytometry. ab32135- intracellular target. Intracellular protocol is recommended for flow cytometry. ab32536- intracellular target. Intracellular protocol is recommended for flow cytometry. ab32360- intracellular target. Intracellular protocol is recommended for flow cytometry. ab40752- intracellular target. Intracellular protocol is recommended for flow cytometry. ab76311- intracellular target. Intracellular protocol is recommended for flow cytometry. ab76429- intracellular target. Intracellular protocol is recommended for flow cytometry. ab80392- Bcl10 antibody [151] (Phycoerythrin) - Does not need a secondary detection antibody. Intracellular target. Intracellular protocol is recommended for flow cytometry. Our protocols are found on https://www.abcam.com/protocols I have attached a protocol for intracellular targets from one of our collaborators (this protocol is also inluded under the link) I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Read More

    Abcam Scientific Support

    Answered on Jan 12 2011

    Question

    I'm using abcam antibody ab32041 for westernblot and immunohistochemistry. Do you also have a suitable blocking peptide to show specifity in immunohistochemistry?

    Read More

    Abcam community

    Verified customer

    Asked on Oct 14 2009

    Answer

    Thank you for your enquiry. Unfortunately, the blocking peptide for this product is not available. I am sorry I can't be of much help in this occasion but if there is anything else that I can help you with, please do not hesitate to contact me.

    Read More

    Abcam Scientific Support

    Answered on Oct 14 2009

    Western blot abreview for Anti-IKK alpha antibody [Y463]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Epithelial, Neutrophils, monocytes)
    Loading amount
    500000 cells
    Specification
    Epithelial, Neutrophils, monocytes
    Gel Running Conditions
    Reduced Denaturing (4-12% Bis-Tris gel)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
    Read More

    Abcam user community

    Verified customer

    Submitted Sep 02 2009

    Immunohistochemistry (Frozen sections) abreview for Anti-IKK alpha antibody [Y463]

    Below Average
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Mouse Tissue sections (skin)
    Specification
    skin
    Fixative
    Paraformaldehyde
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 6%
    Read More

    Abcam user community

    Verified customer

    Submitted Apr 24 2007

    Question

    What is the concentration of this antibody?

    Read More

    Abcam community

    Verified customer

    Asked on Apr 19 2006

    Answer

    Thank you for your enquiry. The concentration for the current lot is 0.25mg/ml. I hope this information helps. Please do not hesitate to contact us if you need anything further.

    Read More

    Abcam Scientific Support

    Answered on Apr 19 2006

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
    For licensing inquiries, please contact partnerships@abcam.com

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