Overview

  • Product name
    Human EGFR In-Cell ELISA Kit
    See all EGFR kits
  • Detection method
    Colorimetric
  • Sample type
    Adherent cells
  • Assay type
    Cell-based (qualitative)
  • Assay time
    5h 10m
  • Assay duration
    Multiple steps standard assay
  • Species reactivity
    Reacts with: Human
  • Product overview

    ab126419 is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in cells. It can be used for measuring the relative amount of EGFR (activated) phosphorylation and screening the effects of various treatments, inhibitors (such as siRNA or chemicals), or activators in cultured Human cell lines. By determining EGFR protein phosphorylation in your experimental model system, you can verify pathway activation in your cell lines without spending excess time and effort in preparing cell lysate and performing an analysis of Western Blot.

    In the EGFR Human In-Cell ELISA kit, cells are seeded into a 96 well tissue culture plate. The cells are fixed after various treatments, inhibitors or activators. After blocking, Anti-Phospho-EGFR (activated) or Anti-EGFR (primary antibody) is pipetted into the wells and incubated. The wells are washed, and HRP-conjugated anti mouse IgG (secondary antibody) is added to the wells. The wells are washed again, a TMB substrate solution is added to the wells and color develops in proportion to the amount of protein. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

  • Tested applications
    Suitable for: In-Cell ELISAmore details
  • Platform
    Microplate

Properties

  • Storage instructions
    Store at -20°C. Please refer to protocols.
  • Components 1 x 96 tests
    HRP-conjugated Anti-Mouse IgG Concentrate 1 x 10µl
    Blocking Buffer Concentrate (5X) 1 x 20ml
    Fixing Solution 1 x 30ml
    Uncoated 96-well Microplate 1 unit
    Mouse Anti-EGFR Concentrate (Item H) 1 x 7µl
    Mouse Anti-Phospho-EGFR (active) Concentrate (Item G) 1 x 7µl
    Quenching Buffer Concentrate (30x) 1 x 2ml
    Stop Solution 1 x 14ml
    TMB One-Step Substrate Reagent 1 x 12ml
    Wash Buffer A Concentrate (20X) 1 x 30ml
    Wash Buffer B Concentrate (20X) 1 x 30ml
  • Research areas
  • Function
    Receptor for EGF, but also for other members of the EGF family, as TGF-alpha, amphiregulin, betacellulin, heparin-binding EGF-like growth factor, GP30 and vaccinia virus growth factor. Is involved in the control of cell growth and differentiation. Phosphorylates MUC1 in breast cancer cells and increases the interaction of MUC1 with SRC and CTNNB1/beta-catenin.
    Isoform 2 may act as an antagonist of EGF action.
  • Tissue specificity
    Ubiquitously expressed. Isoform 2 is also expressed in ovarian cancers.
  • Involvement in disease
    Defects in EGFR are associated with lung cancer (LNCR) [MIM:211980].
  • Sequence similarities
    Belongs to the protein kinase superfamily. Tyr protein kinase family. EGF receptor subfamily.
    Contains 1 protein kinase domain.
  • Post-translational
    modifications
    Phosphorylation of Ser-695 is partial and occurs only if Thr-693 is phosphorylated.
    Monoubiquitinated and polyubiquitinated upon EGF stimulation; which does not affect tyrosine kinase activity or signaling capacity but may play a role in lysosomal targeting. Polyubiquitin linkage is mainly through 'Lys-63', but linkage through 'Lys-48', 'Lys-11' and 'Lys-29' also occur.
  • Cellular localization
    Secreted and Cell membrane. Endoplasmic reticulum membrane. Golgi apparatus membrane. Nucleus membrane. Endosome. In response to EGF, translocated from the cell membrane to the nucleus via Golgi and ER. Co-localizes with TNK2 on the endosomes.
  • Information by UniProt
  • Alternative names
    • 9030024J15Rik
    • AI552599
    • Avian erythroblastic leukemia viral (v erb b) oncogene homolog
    • Avian erythroblastic leukemia viral (verbb) oncogene homolog
    • Cell growth inhibiting protein 40
    • Cell proliferation inducing protein 61
    • EGF R
    • EGFR
    • EGFR_HUMAN
    • Epidermal growth factor receptor
    • Epidermal growth factor receptor (avian erythroblastic leukemia viral (v erb b) oncogene homolog)
    • Epidermal growth factor receptor (erythroblastic leukemia viral (v erb b) oncogene homolog avian)
    • Erbb
    • erbb 1
    • Erbb1
    • Erythroblastic leukemia viral (v erb-b) oncogene homolog
    • HER1
    • mENA
    • Oncogene ERBB
    • OTTMUSP00000005385
    • OTTMUSP00000005386
    • PIG61
    • Proto oncogene c ErbB 1
    • Proto-oncogene c-ErbB-1
    • Receptor tyrosine protein kinase ErbB 1
    • Receptor tyrosine protein kinase ErbB1
    • Receptor tyrosine-protein kinase ErbB-1
    • RP23-295E4.1
    • Urogastrone
    • wa2
    • Wa5
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab126419 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
In-Cell ELISA Use at an assay dependent concentration.

Images

  • A431 cells were stimulated by different concentrations of EGF for 20 min at 37°C.

  • Western blot analysis of extracts from 100 ng/ml hEGF treated A431 cells. Phospho-EGFR (activated) and EGFR antibodies were used in both detection assays.
  • A431 cells were stimulated by different concentrations of EGF for 20 min at 37°C.

Protocols

References

This product has been referenced in:
  • El-Husseiny WM  et al. Synthesis, antitumour and antioxidant activities of novel a,ß-unsaturated ketones and related heterocyclic analogues: EGFR inhibition and molecular modelling study. J Enzyme Inhib Med Chem 33:507-518 (2018). Read more (PubMed: 29455554) »
See 1 Publication for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question
Answer

The lab let me know the following:

While we cannot say with 100% certainty that this antibody will not cross react with phosphorylated tyrosines on any other molecules besides EGFR, we can predict with pretty high confidence that the antibody will most likely only recognizes phosphorylated tyrosine on EGFR. The evidence to support this is:

The antibody is monoclonal (so it is highly specific for a single epitope on EGFR)
EGFR was the immunogen used to create the antibody
Western blotting shows only a single band detected, ˜180 kDa which is the estimated molecular weight of EGFR. The blot image can be found on the datasheet for the kit: https://www.abcam.com/egfr-human-in-cell-elisa-kit-ab126419.html




I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

Read More

Answer

The anti-phospho antibody in the EGFR ELISA kit is a single monoclonal, not a cocktail of several clones. Although it is not available separately, I did find another antibody that may be of interest, Anti-Phosphoserine/threonine/tyrosine, ab15556.

Click here (or use the following: https://www.abcam.com/index.html?datasheet=15556).

Please let me know if you have any questions.

Read More

Answer

Thank you for your inquiry.

The lab has confirmed that the phospho EGFR antibody is monoclonal, was purified by protein A chromatography, the concentration is not available (it is not necessary to perform the assay), and the buffer is an aqueous buffer solution containing BSA, glycerol, and sodium azide.

I hope this information helps. Please contact us with any other questions.

Read More

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

Sign up