Anti-EGFR Affibody® Molecule Agarose Immobilized (ab36039)
Overview
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Product name
Anti-EGFR Affibody® Molecule Agarose Immobilized
See all EGFR affibody® molecule -
Conjugation notes
There is no decrease in performance of the agarose immobilized Affibody® molecule after storage for 2 weeks at +37 degrees C. -
Specificity
This product binds to human EGFR. This anti-EGFR Affibody® molecule is a specific affinity ligand selected against the extracellular domain of EGFR. -
Tested applications
Suitable for: IP, ICC/IF, Flow Cytmore details -
Species reactivity
Reacts with: Human -
Immunogen
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General notes
This 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 6kDa, 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
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C. Do Not Freeze. -
Storage buffer
pH: 7.50
Preservative: 0.02% Sodium azide
Constituents: 0.87% Sodium chloride, 0.328% Sodium phosphate
Supplied as 50% slurry in buffer solution:Preservative: 0.02% Sodium AzideConstituents: 20mM Sodium phosphate, 150mM Sodium chloride, pH 7.5 -
Concentration information loading... -
Purification notes
The purity of this product is >98% as determined by SDS-PAGE and RP-HPLC analysis. -
Research areas
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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
modificationsPhosphorylation 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
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Alternative names
- Avian erythroblastic leukemia viral (v erb b) oncogene homolog
- Cell growth inhibiting protein 40
- Cell proliferation inducing protein 61
see all -
Database links
- Entrez Gene: 1956 Human
- Omim: 131550 Human
- SwissProt: P00533 Human
- Unigene: 488293 Human
- Unigene: 605083 Human
Images
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Immunoprecipitation - Anti-EGFR Affibody® Molecule Agarose Immobilized (ab36039)
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.
