Overview

  • Product name

    Mouse IkappaB alpha ELISA Kit
  • Detection method

    Colorimetric
  • Precision

    Intra-assay
    Sample n Mean SD CV%
    cell extract 5 6.5%
    Inter-assay
    Sample n Mean SD CV%
    cell extract 3 10%
  • Sample type

    Cell culture extracts, Tissue Extracts
  • Assay type

    Sandwich (quantitative)
  • Sensitivity

    14.7 pg/ml
  • Range

    46.875 pg/ml - 3000 pg/ml
  • Recovery

    Sample specific recovery
    Sample type Average % Range
    Cell culture media 90.9 89.7% - 92.7%

  • Assay time

    1h 30m
  • Assay duration

    One step assay
  • Species reactivity

    Reacts with: Mouse
  • Product overview

    Abcam’s IkappaB alpha in vitro SimpleStep ELISA® (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of IkappaB alpha protein in mouse cell and tissue extract samples.

    The SimpleStep ELISA® employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.

  • Notes

    IkappaB alpha 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, IkappaB alpha becomes phosphorylated promoting ubiquitination and degradation, enabling the dimeric RELA to translocate to the nucleus and activate transcription.

  • Tested applications

    Suitable for: Sandwich ELISAmore details
  • Platform

    Pre-coated microplate (12 x 8 well strips)

Properties

  • Storage instructions

    Store at +4°C. Please refer to protocols.
  • Components 1 x 96 tests
    10X Mouse IkappaB alpha Detector Antibody 1 x 600µl
    10X Wash Buffer PT (ab206977) 1 x 20ml
    50X Cell Extraction Enhancer Solution (ab193971) 1 x 1ml
    5X Cell Extraction Buffer PTR (ab193970) 1 x 10ml
    Antibody Diluent 4BR 1 x 6ml
    Mouse IkappaB alpha Lyophilized Recombinant Protein 2 vials
    Mouse IkappaB alpha Capture Antibody (lyophilized) 1 vial
    Plate Seals 1 unit
    Sample Diluent NS (ab193972) 1 x 12ml
    SimpleStep Pre-Coated 96-Well Microplate (ab206978) 1 unit
    Stop Solution 1 x 12ml
    TMB Development Solution 1 x 12ml
  • Research areas

  • 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 tranlocate to the nucleus and activate transcription.
  • Involvement in disease

    Defects in NFKBIA are the cause of ectodermal dysplasia anhidrotic with T-cell immunodeficiency autosomal dominant (ADEDAID) [MIM:612132]. Ectodermal dysplasia defines a heterogeneous group of disorders due to abnormal development of two or more ectodermal structures. ADEDAID is an ectodermal dysplasia associated with decreased production of pro-inflammatory cytokines and certain interferons, rendering patients susceptible to infection.
  • 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.
    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 polyubiquitinatin leads to protein degredation. 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 interefers with NFKBIA degradation and impairs subsequenbt 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
  • Alternative names

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

Applications

Our Abpromise guarantee covers the use of ab206308 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
Sandwich ELISA Use at an assay dependent concentration.

Images

  • SimpleStep ELISA technology allows the formation of the antibody-antigen complex in one single step, reducing assay time to 90 minutes. Add samples or standards and antibody mix to wells all at once, incubate, wash, and add your final substrate. See protocol for a detailed step-by-step guide.

     

  • Background-subtracted data values (mean +/- SD) are graphed.

  • The concentrations of IkappaB alpha were measured in duplicates, interpolated from the IkappaB alpha standard curves and corrected for sample dilution. Note that 1X Diluted A20 cell extract samples were at 50 µg/mL. The interpolated, dilution factor-corrected values are plotted in pg of IkappaB alpha per µg of total protein (mean +/- SD, n=2).

  • Cell extracts were prepared from untreated mouse A20, rat H9C2 and rat H4II2 cells, or mouse NIH/3T3 cells treated for 20 minutes with 20 ng/mL TNF-alpha or mock-treated. The concentrations of IkappaB alpha were measured in three dilutions in duplicates, interpolated from the IkappaB alpha standard curves and corrected for sample dilution. Note that 1X Diluted A20 cell extract samples were at 50 µg/mL. Note that 1X Diluted NIH/3T3 cell extract samples were at 25 µg/mL. Note that 1X Diluted H9C2 and H4IIE cell extract samples were at 200 µg/mL The interpolated, dilution factor-corrected values are plotted in pg of IkappaB alpha per µg of total protein (mean +/- SD, n=2). As expected, treatment of NIH/3T3 cells with TNF-alpha reduces IkappaB alpha concentration.

Protocols

References

ab206308 has not yet been referenced specifically in any publications.

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