Overview

  • Product name
    Anti-IKB alpha antibody [E130] - BSA and Azide free
    See all IKB alpha primary antibodies
  • Description
    Rabbit monoclonal [E130] to IKB alpha - BSA and Azide free
  • Host species
    Rabbit
  • Tested applications
    Suitable for: Flow Cyt, WB, IP, ICC/IF, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Hamster, Human
    Predicted to work with: Cow, Pig
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) corresponding to Human IKB alpha.

  • Positive control
    • Hela cell lysate and human prostate carcinoma tissue.
  • General notes

    The formulation and the concentration of this product is compatible for metal-conjugation for mass cytometry (CyTOF®).

    ab215972 is a PBS-only buffer formulated version of ab32518, containing no BSA or sodium azide, ideal for antibody labeling. Please refer to ab32518 for information on protocols, dilutions, and image data.

    Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    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 ab215972 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
Flow Cyt Use at an assay dependent concentration.
WB Use at an assay dependent concentration. Detects a band of approximately 35 kDa (predicted molecular weight: 36 kDa).
IP Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.

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
  • 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

  • This WB data was generated using the same anti-IKB alpha antibody clone, E130, in a different buffer formulation (cat# ab32518).

    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 - ab32518 observed at 38 kDa. Red - loading control, ab18058, observed at 130 kDa.

    ab32518 was shown to specifically react with IKB alpha in wild-type HAP1 cells. No band was observed when IKB alpha knockout samples were tested. Wild-type and IKB alpha knockout samples were subjected to SDS-PAGE. Ab32518 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/10,000 dilution and 1/20,000 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 1/20,000 dilution for 1 hour at room temperature before imaging.

  • Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling IKB alpha with purified ab32518 at 1/20 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32518).

  • Immunofluorescence staining of HeLa cells with purified ab32518 at a working dilution of 1 in 50, counter-stained with DAPI. The secondary antibody was ab150077, Alexa Fluor® 488 goat anti rabbit, used at a dilution of 1 in 500. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative control is shown in bottom right hand panel - for the negative control, purified ab32518 was used at a dilution of 1/50 followed by ab150120, Alexa Fluor® 594 goat anti-mouse antibody at a dilution of 1/500.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32518).

  • Immunohistochemical staining of paraffin embedded rat kidney with purified ab32518 at a working dilution of 1 in 100. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32518).

  • ab32518 (purified) at 1/20 immunoprecipitating IKB alpha in HeLa cell lysate (Lane 1). For western blotting a HRP-conjugated goat anti-rabbit IgG was used as the secondary antibody (1/1000).

    Blocking buffer and concentration: 5% NFDM/TBST.

    Diluting buffer and concentration: 5% NFDM /TBST.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32518).

  • Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using unpurified ab32518 at 1/50 dilution.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32518).

  • Unpurified ab32518 used to immunoprecipitate IKB alpha from human HeLa whole cell lysate. The antibody was further used to Western blot the protein.

    Lane 1 IKB alpha IP

    Lane 2 Control immunoprecipitate

    Lane 3 Input (20%)

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32518).

  • Unpurified ab32518 used to immunoprecipitate IKB alpha from rat PC12 whole cell lysate. The antibody was further used to Western blot the protein.

    Lane 1 IKB alpha IP

    Lane 2 Control immunoprecipitate

    Lane 3 Input (20%)

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32518).

  • Unpurified ab32518 at 1/100 staining mouse kidney tissue sections by IHC-P. The tissue was paraformaldehyde fixed and a heat mediated antigen retrieval step in citrate buffer was performed before the tissue was blocked and incubated with the antibody for 1 hour. An HRP conjugated goat anti-rabbit antibody was used as the secondary.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32518).

  • ICC/IF image of unpurified ab32518 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32518, 1/1000 dilution) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32518).

  • Unpurified ab32518 staining IkBα/&beta in RAW 264.7 cells treated with FK506 (ab120223), by ICC/IF. Decrease in IkBα/&beta expression correlates with increased concentration of FK506, as described in literature.
    The cells were incubated at 37°C for 3h in media containing different concentrations of ab120223 (FK506) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab32518 (1/100 dilution) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight® 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32518).

  • This IHC data was generated using the same anti-IKB alpha antibody clone, E130, in a different buffer formulation (cat# ab32518).

    Immunohistochemical staining of paraffin embedded human stomach with purified ab32518 at a working dilution of 1 in 100. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

References

This product has been referenced in:
  • Clift D  et al. A Method for the Acute and Rapid Degradation of Endogenous Proteins. Cell 171:1692-1706.e18 (2017). Read more (PubMed: 29153837) »
  • Nakayama T  et al. Mutations in PYCR2, Encoding Pyrroline-5-Carboxylate Reductase 2, Cause Microcephaly and Hypomyelination. Am J Hum Genet 96:709-19 (2015). WB ; Human . Read more (PubMed: 25865492) »
See all 25 Publications for this product

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