Overview

Description

  • Nature
    Recombinant
  • Source
    Insect cells
  • Amino Acid Sequence
    • Species
      Human
    • Amino acids
      695 to 1210
    • Tags
      GST tag N-Terminus

Specifications

Our Abpromise guarantee covers the use of ab89746 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

  • Biological activity
    The Specific activity of ab89746 was determined to be 60 nmol/min/mg.
  • Applications

    Western blot

    Functional Studies

  • Form
    Liquid
  • Additional notes

    ab204877 (Poly (4:1 Glu, Tyr) peptide) can be utilized as a substrate for assessing kinase activity

  • Concentration information loading...

Preparation and Storage

  • Stability and Storage

    Shipped on dry ice. Upon delivery aliquot and store at -80ºC. Avoid freeze / thaw cycles.

    pH: 7.50
    Constituents: 0.0038% EGTA, 0.00174% PMSF, 0.00385% DTT, 0.79% Tris HCl, 0.00292% EDTA, 25% Glycerol, 0.87% Sodium chloride

    This product is an active protein and may elicit a biological response in vivo, handle with caution.

General Info

  • Alternative names
    • Avian erythroblastic leukemia viral (v erb b) 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)
    • erb-b2 receptor tyrosine kinase 1
    • ERBB
    • ERBB1
    • Errp
    • HER1
    • mENA
    • NISBD2
    • Oncogen ERBB
    • PIG61
    • Proto-oncogene c-ErbB-1
    • Receptor tyrosine protein kinase ErbB 1
    • Receptor tyrosine-protein kinase ErbB-1
    • SA7
    • Species antigen 7
    • Urogastrone
    • v-erb-b Avian erythroblastic leukemia viral oncogen homolog
    • wa2
    • Wa5
    see all
  • Function
    Receptor tyrosine kinase binding ligands of the EGF family and activating several signaling cascades to convert extracellular cues into appropriate cellular responses. Known ligands include EGF, TGFA/TGF-alpha, amphiregulin, epigen/EPGN, BTC/betacellulin, epiregulin/EREG and HBEGF/heparin-binding EGF. Ligand binding triggers receptor homo- and/or heterodimerization and autophosphorylation on key cytoplasmic residues. The phosphorylated receptor recruits adapter proteins like GRB2 which in turn activates complex downstream signaling cascades. Activates at least 4 major downstream signaling cascades including the RAS-RAF-MEK-ERK, PI3 kinase-AKT, PLCgamma-PKC and STATs modules. May also activate the NF-kappa-B signaling cascade. Also directly phosphorylates other proteins like RGS16, activating its GTPase activity and probably coupling the EGF receptor signaling to the G protein-coupled receptor signaling. Also phosphorylates MUC1 and increases its interaction 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
    Lung cancer
    Inflammatory skin and bowel disease, neonatal, 2
  • Sequence similarities
    Belongs to the protein kinase superfamily. Tyr protein kinase family. EGF receptor subfamily.
    Contains 1 protein kinase domain.
  • Post-translational
    modifications
    Phosphorylation at Ser-695 is partial and occurs only if Thr-693 is phosphorylated. Phosphorylation at Thr-678 and Thr-693 by PRKD1 inhibits EGF-induced MAPK8/JNK1 activation. Dephosphorylation by PTPRJ prevents endocytosis and stabilizes the receptor at the plasma membrane. Autophosphorylation at Tyr-1197 is stimulated by methylation at Arg-1199 and enhances interaction with PTPN6. Autophosphorylation at Tyr-1092 and/or Tyr-1110 recruits STAT3. Dephosphorylated by PTPN1 and PTPN2.
    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 occurs. Deubiquitination by OTUD7B prevents degradation. Ubiquitinated by RNF115 and RNF126.
    Methylated. Methylation at Arg-1199 by PRMT5 stimulates phosphorylation at Tyr-1197.
  • Cellular localization
    Secreted and Cell membrane. Endoplasmic reticulum membrane. Golgi apparatus membrane. Nucleus membrane. Endosome. Endosome membrane. Nucleus. In response to EGF, translocated from the cell membrane to the nucleus via Golgi and ER. Endocytosed upon activation by ligand. Colocalized with GPER1 in the nucleus of estrogen agonist-induced cancer-associated fibroblasts (CAF).
  • Information by UniProt

Images

  • SDS-PAGE showing ab89746 at approximately 89kDa.
  • Kinase Assay demonstrating specific activity of ab89746.

References

ab89746 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Answer

If your sample is 90% pure EGFR, then a concentration of 2 ug/ml will give you a signal. You may find that less is sufficient, so I recommend testing a range, such as 0.125, 0.5, and 2 ug/ml. The note in the protocol recommending 20 ug/ml refers to total protein content of samples such as cell lysates and tissue homogenates which contain other proteins in addition to EGFR. Please let me know if you have other questions.

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Answer

That is correct, amino acid range aa 695-1210 is a cytoplasmic (i.e., intracellular) domain. The numbering refers to the sequence at the following UniProt entry, and a list of the domains and the amino acid ranges that constitute these domains is about a quarter of the way down the page of this entry, in the "Sequence annotation (Features)" section.

http://www.uniprot.org/uniprot/P00533

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Answer

No, the expression host, virus-infected insect cells, will have no bearing on the reactivity in this case. I recommend choosing an antibody that is known to react with EGFR in its native conformation, as demonstrated, for instance, by immunocytochemistry or immunohistochemistry or, ideally, ELISA of non-denatured cell or tissue lysates.

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Answer

It is very likely that ab89746 can be used in a direct ELISA, assuming you try to detect it with an antibody that is raised against an amino acid sequence that is present in ab89746, which is a recombinant fragment of full-length EGFR, from amino acid 695 to 1210. However, the protein has not been tested in a direct ELISA, to our knowledge, and the application is not covered by our Abpromise guarantee for ab89746.

The protein will differ from EGFR from A431 cells, whether endogenous or full-length recombinant, insofar as it is a fragment, and is expressed in a Baculovirus-infected Sf9 insect cell expression system. Please note also that ab89746 has an N-terminal GST tag, which may hinder recognition by antibodies raised against a peptide containing the ab89746 N-terminus amino acid, aa695.

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Question
Answer

Thank you for your phone call.


DISCOUNT CODE: xxx
Expiration date: xxx

I am very pleased to hear you would like to accept our offer and test ab89746 in sELISA. This code will give you 1 freePROTEIN before the expiration date. To redeem this offer, please submit an Abreview for sELISAand include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: https://www.abcam.com/collaborationdiscount.

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Answer

I have received more information from my colleague which I would like to share with you. Unfortunately they have recommended not using ab89746 because ligand receptor association cannot be achieved extracellularly due to the hydrophilic nature of the external surface of protein. The ligand and receptor binding seems impossible extraccellularly to capture because precipitation occurs due to the hydrophilic nature. We were only able to capture the conformational change of the ligand binding to the receptor intracellularly. I hope this will give you more insight while choosing the product. Should you have any other question please do not hesitate to ask.

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Answer

Thank you for contacting us. We have not tested these products in Biocore (surface plasmon resonance) experiments. Theoretically these products should work however the conditions have to be optimized by end user. We can recommend coating ligand (in this case EGF) which sometime works better instead of sEGFR. I hope this information is nevertheless helpful. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this protein. In answer to your question, I am contacting our source to obtain the testing procedure for you. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund. I would like to investigate this particular case further for you, and also obtain more information for our quality records. In order to proceed with this, I would appreciate if you could provide a few details: 1. Please confirm how has the protein been stored? 2. Please confirm the purchase order number and date of purchase. 3. Have you tried your protocol with other positive control samples? What were the results? 4. What procedure are you using? Please provide more details. 5. Have you run some of the protein on an SDS gel with coomassie staining to check if it is degraded? I would appreciate if you could also provide any data or images which would help us to assess the results. Thank you for your time and cooperation. I look forward to hearing from you with the requested details.

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