Overview

  • Product name
    I kappaB alpha (pS32/36) ELISA Kit
  • Detection method
    Colorimetric
  • Precision
    Intra-assay
    Sample n Mean SD CV%
    HeLa extract 6 2.8%
    Inter-assay
    Sample n Mean SD CV%
    HeLa extract 3 4.4%
  • Sample type
    Cell Lysate, Tissue Homogenate
  • Assay type
    Semi-quantitative
  • Sensitivity
    0.06 ng/ml
  • Range
    0.06 ng/ml - 6 ng/ml
  • Assay time
    1h 30m
  • Assay duration
    One step assay
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Mouse, Rat
  • Product overview

    Abcam’s I?Bα (pS32/36) in vitro SimpleStep ELISA™ (Enzyme-Linked Immunosorbent Assay) kit is designed for the semi-quantitative measurement of I?Bα (pS32/36) protein in Human cells.

    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.

  • Tested applications
    Suitable for: Sandwich ELISAmore details
  • Platform
    Microplate

Properties

  • Storage instructions
    Store at +4°C. Please refer to protocols.
  • Components 1 x 96 tests
    10X Wash Buffer PT 1 x 15ml
    50X Cell Extraction Enhancer Solution 1 x 1ml
    5X Cell Extraction Buffer PTR (ab193970) 1 x 12ml
    I kappaB alpha (pS32/36) Capture Antibody 1 x 3ml
    I kappaB alpha (pS32/36) Detector Antibody 1 x 3ml
    Lyophilized I kappaB alpha Control Lysate 1 vial
    Plate Seal 1 unit
    SimpleStep Pre-Coated 96-Well Microplate (ab206978) 1 unit
    Stop Solution 1 x 12ml
    TMB Substrate 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

Associated products

Applications

Our Abpromise guarantee covers the use of ab176643 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

  • Example of a typical IĸBα (pS32/36) cell lysate dilution series.  Background-subtracted data values (mean +/- SD) are graphed.

  • Example of a typical IĸBα (pS32/36) recombinant protein standard curve. The proportion of total protein that is phosphorylated is unknown - data is indicative only.  Background-subtracted data values (mean +/- SD) are graphed.

  • Linearity of dilution in representative sample matrices. Cellular lysates were prepared at 3 concentrations in common media containing 1 x Cell Extraction Buffer PTR. Data from duplicate measurements of IĸBα (pS32/36) are normalized and plotted.

  • Cell line analysis for Total IĸBα from 150 µg/mL preparations of cell extracts. Data from triplicate measurements (mean +/- SD) are plotted and compared to 1X Cell Extraction Buffer PTR (zero).

  • Induction of IĸBα (pS32/36) phosphorylation in HeLa cells in response to TNFα treatment. HeLa cells were cultured in 96-well tissue culture plates, and treated (10 min) with a dose-range of TNFα before cell lysis. Data from quadruplicate measurements of IĸBα (pS32/36) are plotted and compared against Total IĸBα protein levels. Comparative IĸBα (pS32/36) and IĸBα (Total) data also shown by Western Blot.

Protocols

References

This product has been referenced in:
  • Kan MH  et al. Pyrrolidine Dithiocarbamate Prevents Neuroinflammation and Cognitive Dysfunction after Endotoxemia in Rats. Front Aging Neurosci 8:175 (2016). Sandwich ELISA ; Rat . Read more (PubMed: 27493629) »
See 1 Publication for this product

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