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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-IKB alpha antibody [EP697] (ab76429)

Reviews (1)Q&A (1)References (15)
Western blot - Anti-IKB alpha antibody [EP697] (ab76429)
  • Western blot - Anti-IKB alpha antibody [EP697] (ab76429)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IKB alpha antibody [EP697] (ab76429)
  • Immunocytochemistry/ Immunofluorescence - Anti-IKB alpha antibody [EP697] (ab76429)
  • Western blot - Anti-IKB alpha antibody [EP697] (ab76429)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IKB alpha antibody [EP697] (ab76429)
  • Western blot - Anti-IKB alpha antibody [EP697] (ab76429)
  • Anti-IKB alpha antibody [EP697] (ab76429)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EP697] to IKB alpha
  • Suitable for: WB, IHC-P, ICC/IF
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Conjugates logo Related conjugates and formulations

Alexa Fluor® 647 Carrier Free

Overview

  • Product name

    Anti-IKB alpha antibody [EP697]
    See all IKB alpha primary antibodies

  • Description

    Rabbit monoclonal [EP697] to IKB alpha

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, IHC-P, ICC/IFmore details
    Unsuitable for: Flow Cyt

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Synthetic peptide within Human IKB alpha (C terminal). The exact sequence is proprietary.

  • Positive control

    • HeLa, PC-12, RAW264.7 and Jurkat cell lysates; human kidney and colon tissue.

  • 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.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab76429 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)
1/500 - 1/10000. Detects a band of approximately 36 kDa (predicted molecular weight: 36 kDa).
IHC-P
1/500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

For unpurified use at 1/100 - 1/250.
ICC/IF
1/250.

For unpurified use at 1/50 - 1/100.
Notes
WB
1/500 - 1/10000. Detects a band of approximately 36 kDa (predicted molecular weight: 36 kDa).
IHC-P
1/500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

For unpurified use at 1/100 - 1/250.
ICC/IF
1/250.

For unpurified use at 1/50 - 1/100.
Application notes
Is unsuitable for Flow Cyt.

Target

  • Function

    Inhibits the activity of dimeric NF-kappa-B/REL complexes by trapping REL dimers in the cytoplasm through masking of their nuclear localization signals. On cellular stimulation by immune and proinflammatory responses, becomes phosphorylated promoting ubiquitination and degradation, enabling the dimeric RELA to translocate to the nucleus and activate transcription.

  • Involvement in disease

    Ectodermal dysplasia, anhidrotic, with T-cell immunodeficiency autosomal dominant

  • Sequence similarities

    Belongs to the NF-kappa-B inhibitor family.
    Contains 5 ANK repeats.

  • Post-translational
    modifications

    Phosphorylated; disables inhibition of NF-kappa-B DNA-binding activity. Phosphorylation at positions 32 and 36 is prerequisite to recognition by UBE2D3 leading to polyubiquitination and subsequent degradation.
    Sumoylated; sumoylation requires the presence of the nuclear import signal. Sumoylation blocks ubiquitination and proteasome-mediated degradation of the protein thereby increasing the protein stability.
    Monoubiquitinated at Lys-21 and/or Lys-22 by UBE2D3. Ubiquitin chain elongation is then performed by CDC34 in cooperation with the SCF(FBXW11) E3 ligase complex, building ubiquitin chains from the UBE2D3-primed NFKBIA-linked ubiquitin. The resulting polyubiquitination leads to protein degradation. Also ubiquitinated by SCF(BTRC) following stimulus-dependent phosphorylation at Ser-32 and Ser-36.
    Deubiquitinated by porcine reproductive and respiratory syndrome virus Nsp2 protein, which thereby interferes with NFKBIA degradation and impairs subsequent NF-kappa-B activation.

  • Cellular localization

    Cytoplasm. Nucleus. Shuttles between the nucleus and the cytoplasm by a nuclear localization signal (NLS) and a CRM1-dependent nuclear export.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • I kappa B alpha antibody
    • I-kappa-B-alpha antibody
    • IkappaBalpha antibody
    • IkB-alpha antibody
    • IKBA antibody
    • IKBA_HUMAN antibody
    • IKBalpha antibody
    • MAD 3 antibody
    • MAD3 antibody
    • Major histocompatibility complex enhancer-binding protein MAD3 antibody
    • NF kappa B inhibitor alpha antibody
    • NF-kappa-B inhibitor alpha antibody
    • NFKBI antibody
    • NFKBIA antibody
    • Nuclear factor of kappa light chain gene enhancer in B cells antibody
    • Nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha antibody
    see all

Images

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

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

    Lane 1 : Jurkat (human acute T cell leukemia) whole cell lysate
    Lane 2 : HeLa (human cervix adenocarcinoma) whole cell lysate

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 36 kDa
    Observed band size: 39 kDa why is the actual band size different from the predicted?



    Blocking/Diluting buffer 5% NFDM /TBST
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IKB alpha antibody [EP697] (ab76429)
    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.

  • Immunocytochemistry/ Immunofluorescence - Anti-IKB alpha antibody [EP697] (ab76429)
    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).

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

    Lane 1 : RAW264.7 (mouse abelson murine leukemia virus-induced tumor) whole cell lysate
    Lane 2 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 36 kDa
    Observed band size: 39 kDa why is the actual band size different from the predicted?



    Blocking/Diluting buffer 5% NFDM /TBST
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IKB alpha antibody [EP697] (ab76429)
    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.

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

    Lane 1 : HeLa cell lysates
    Lane 2 : Jurkat cell lysates

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : goat anti-rabbit HRP at 1/1000 dilution

    Predicted band size: 36 kDa
    Observed band size: 36 kDa

  • Anti-IKB alpha antibody [EP697] (ab76429)
    Anti-IKB alpha antibody [EP697] (ab76429)

References (15)

Publishing research using ab76429? Please let us know so that we can cite the reference in this datasheet.

ab76429 has been referenced in 15 publications.

  • Yuan W  et al. LncRNA PVT1 accelerates LPS-induced septic acute kidney injury through targeting miR-17-5p and regulating NF-?B pathway. Int Urol Nephrol N/A:N/A (2021). PubMed: 34089461
  • Zhu Z  et al. Golgi phosphoprotein 3 promotes the proliferation of gallbladder carcinoma cells via regulation of the NLRP3 inflammasome. Oncol Rep 45:N/A (2021). PubMed: 33907835
  • Li S  et al. Schisandrin B inhibits epithelial-mesenchymal transition and stemness of large-cell lung cancer cells and tumorigenesis in xenografts via inhibiting the NF-?B and p38 MAPK signaling pathways. Oncol Rep 45:N/A (2021). PubMed: 33907830
  • Wang Z  et al. Hypoxia-induced PLOD1 overexpression contributes to the malignant phenotype of glioblastoma via NF-?B signaling. Oncogene N/A:N/A (2021). PubMed: 33420370
  • Wang SH  et al. Insulin-like growth factor binding protein 3 promotes radiosensitivity of oral squamous cell carcinoma cells via positive feedback on NF-?B/IL-6/ROS signaling. J Exp Clin Cancer Res 40:95 (2021). PubMed: 33712045
View all Publications for this product

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1-2 of 2 Abreviews or Q&A

Western blot abreview for Anti-IKB alpha antibody [EP697]

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Deer mouse Tissue lysate - whole (Liver)
Gel Running Conditions
Non-reduced Denaturing
Loading amount
20 µg
Specification
Liver
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

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Verified customer

Submitted Oct 28 2019

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

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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.

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