Anti-EGFR Affibody® Molecule Agarose Immobilized (ab36039)

Overview

  • Product nameAnti-EGFR Affibody® Molecule Agarose Immobilized
    See all EGFR affibody® molecule
  • Conjugation notesThere is no decrease in performance of the agarose immobilized Affibody® molecule after storage for 2 weeks at +37 degrees C.
  • SpecificityThis product binds to human EGFR. This anti-EGFR Affibody® molecule is a specific affinity ligand selected against the extracellular domain of EGFR.
  • Tested applicationsSuitable for: IP, ICC/IF, Flow Cytmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    -

  • General notesThis product is a recombinant protein produced in E.coli.


    What are Affibody Molecules?
    Affibody® affinity ligands are unique research reagents, produced using innovative protein-engineering technologies. They are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of Protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This scaffold has excellent features as an affinity ligand and can be designed to bind with high affinity to any given target protein. The domain consists of 58 amino acids, 13 of which are randomized to generate Affibody® libraries with a large number of ligand variants. Thus, the libraries consist of a multitude of protein ligands with an identical backbone and variable surface-binding properties. In function, Affibody® Molecules mimic monoclonal antibodies. Compared to antibodies, the most striking dissimilarity of Affibody® Molecules is the small size. Affibody® Molecules have a molecular weight of 14kDa, compared to the molecular weight of antibodies, which is 150kDa. In spite of its small size, the binding site of Affibody® Molecules is similar to that of an antibody. The advantages of Affibody® Molcules over antibodies are: -their small size -the simple structure of the molecules -its robust physical properties; able to withstand a broad range of analytical conditions, including extreme pH and elevated temperature -its ability to fold correctly intracellularly -the fast and cost effective production in bacteria -the potential to couple Affibody® Molecules in multimeric constructs Affibody® Molecules have highly competitive properties for applications within affinity purification, sample preparation, protein detection and in vitro diagnostics.

Properties

  • FormLiquid
  • Storage instructionsShipped at 4°C. Store at +4°C. Do Not Freeze.
  • Storage bufferSupplied as 50% slurry in buffer solution:
    Preservative: 0.02% Sodium Azide
    Constituents: 20mM Sodium phosphate, 150mM Sodium chloride, pH 7.5
  • Concentration information loading...
  • Purification notesThe purity of this product is >98% as determined by SDS-PAGE and RP-HPLC analysis.
  • Affibody® molecule notesWhat are Affibody Molecules?
    Affibody® affinity ligands are unique research reagents, produced using innovative protein-engineering technologies. They are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of Protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This scaffold has excellent features as an affinity ligand and can be designed to bind with high affinity to any given target protein. The domain consists of 58 amino acids, 13 of which are randomized to generate Affibody® libraries with a large number of ligand variants. Thus, the libraries consist of a multitude of protein ligands with an identical backbone and variable surface-binding properties. In function, Affibody® Molecules mimic monoclonal antibodies. Compared to antibodies, the most striking dissimilarity of Affibody® Molecules is the small size. Affibody® Molecules have a molecular weight of 14kDa, compared to the molecular weight of antibodies, which is 150kDa. In spite of its small size, the binding site of Affibody® Molecules is similar to that of an antibody. The advantages of Affibody® Molcules over antibodies are: -their small size -the simple structure of the molecules -its robust physical properties; able to withstand a broad range of analytical conditions, including extreme pH and elevated temperature -its ability to fold correctly intracellularly -the fast and cost effective production in bacteria -the potential to couple Affibody® Molecules in multimeric constructs Affibody® Molecules have highly competitive properties for applications within affinity purification, sample preparation, protein detection and in vitro diagnostics.
  • Research areas
  • FunctionReceptor 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 specificityUbiquitously expressed. Isoform 2 is also expressed in ovarian cancers.
  • Involvement in diseaseLung cancer
    Inflammatory skin and bowel disease, neonatal, 2
  • Sequence similaritiesBelongs 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 localizationSecreted 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
  • 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
  • Database links

Applications

Our Abpromise guarantee covers the use of ab36039 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
IP Use at an assay dependent dilution. This molecule can be used for immunoprecipitation of EGFR from complex mixes of proteins.
ICC/IF Use at an assay dependent dilution.
Flow Cyt Use at an assay dependent dilution.

Anti-EGFR Affibody® Molecule Agarose Immobilized images

  • Results

    Cell extracts were prepared from high EGFR expressing human squamos carcinoma cell line A431 , from low EFGR expressing SH-SY5Y (human neuroblastoma) cells and from the EGFR negative RAMOS (human B-cell lymphoma) cell line. The three extracts were incubated with agarose immobilized Anti-EGFR Affibody® molecule for 2 hours. After incubation, the unbound proteins were washed away and the bound protein was eluted and separated with SDS-PAGE and blotted onto a PVDF filter. The filter was stained with an antibody against full length EGFR, approximately 1 40 kDa.

    A single protein with MW of approximately 40 kDa was precipitated from A431 and SH-SY5Y cells but not from RAMOS cells, as shown in figure . With increased amount of A431 proteins, the 40 kDa EGFR band got more intense on the Western blot (lanes -3). The EGFR band was also detected in A431 cell extracts prior to precipitation together with bands of lower MW that were not present after precipitation (lane 4). SH-SY5Y cells also express EGFR but to a lower level than A431 , as shown by a relatively weak EGFR band (lane 5). There were no EGFR detected in SH-SY5Y cell extracts prior to precipitation (lane 6) and there were no EFGR precipitated from RAMOS cells or present in the cell extracts (lanes 7-8).

    In summary, the Anti-EGFR Affibody® molecule efficiently precipitates EGFR from a complex protein mix. When performing immunoprecipitation experiments with antibodies, there is often a problem with cross reaction between the enzyme conjugated second step reagent and the precipitating antibody but this type of cross reaction is elegantly avoided using an Affibody® molecule as the precipitating reagent.

References for Anti-EGFR Affibody® Molecule Agarose Immobilized (ab36039)

ab36039 has not yet been referenced specifically in any publications.

Product Wall

This product, Anti-EGFR Affibody® Molecule Agarose Immobilized, is not an antibody, it is a small ligand that mimics an anti-EGFR antibody. It does not dissociate from the agarose during boiling of the agarose. Even if it did, it is much smaller than t...

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Thanks you for contacting us. The developer of the EGFR Affibody agarose suggests that pre-clearing the lysate may not be necessary. If you do suspect that you are pulling down other proteins in addition to EGFR, and you feel that pre-clearing is neces...

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Thank you for contacting us. The laboratory that developed this product has confirmed: "The product does not contain any Protein A or Protein G. The Anti-EGFR Affibody® Molecule has no affinity for IgG. As you say it is very unlikely that any IgG wo...

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