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.
  • General notes

    A trial size is available to purchase for this antibody.

     

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

    This product is a recombinant rabbit monoclonal antibody.

Properties

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.
Flow Cyt 1/50.

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

IP 1/50.
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.
  • Information by UniProt
  • Database links
  • 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

  • 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.

  • 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.

  • 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.

  • 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?

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

References

This product has been referenced in:
  • Zhang C  et al. Activation of TNF-a/NF-?B axis enhances CRL4BDCAF11E3 ligase activity and regulates cell cycle progression in human osteosarcoma cells. Mol Oncol N/A:N/A (2018). WB . Read more (PubMed: 29377600) »
  • Li J  et al. Emodin attenuates titanium particle-induced osteolysis and RANKL-mediated osteoclastogenesis through the suppression of IKK phosphorylation. Mol Immunol 96:8-18 (2018). Read more (PubMed: 29455094) »
See all 14 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
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

Abcam user community

Verified customer

Submitted Jul 31 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
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

Abcam user community

Verified customer

Submitted Jun 08 2012

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

Abcam user community

Verified customer

Submitted Dec 14 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

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 guarantees this product to work in the species/application used in this Abreview.
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

Abcam user community

Verified customer

Submitted Sep 02 2009

Abcam has not validated the combination of species/application used in this Abreview.
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%

Abcam user community

Verified customer

Submitted Apr 24 2007

Question
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

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Sign up