Key features and details
- Sample type: Adherent cells, Suspension cells
- Detection method: Colorimetric
- Assay type: Cell-based (quantitative)
- Reacts with: Mouse, Rat, Human
Product namePhospho-ErbB2 / HER2 (Y1248) + Total In-Cell ELISA Kit
See all ErbB2 / HER2 kits
Sample typeAdherent cells, Suspension cells
Assay typeCell-based (quantitative)
Assay durationMultiple steps standard assay
Species reactivityReacts with: Mouse, Rat, Human
Phospho-ErbB2 / HER2 (Y1248) + Total In-Cell ELISA Kit (ab207465) provides a simple, efficient, cell-based method to monitor proteins activated by phosphorylation. The kit is designed specifically to quantify phosphorylated ErbB-2 and/or total ErbB2 / HER2. Cells are cultured in 96-well plates and stimulated to induce the pathway of interest. Following stimulation, the cells are rapidly fixed to preserve activation-specific protein modifications. Each well is then incubated with a primary antibody that recognizes either phosphorylated ErbB2 / HER2 or total ErbB2 / HER2. Subsequent incubation with secondary HRP-conjugated antibody and developing solution provides an easily quantified colorimetric readout. The relative number of cells in each well is then determined using the provided Crystal Violet solution. The 96-well plate format is suitable for high-throughput screening applications.
The Phospho-ErbB2 / HER2 (Y1248) + Total In-Cell ELISA Kit (ab207465) contains 96-well plates and two primary antibodies. The phospho-ErbB2 / HER2 antibody was raised in rabbit against a synthetic phospho-peptide corresponding to the sequence surrounding Tyr1248 of human ErbB2 / HER2. This antibody recognizes only phosphorylated ErbB2 / HER2. The total-ErbB2 / HER2 antibody recognizes ErbB2 / HER2 regardless of its phosphorylation state. The kit can be used to study phosphorylated ERbB-2 relative to cell number or to determine ErbB-2 phosphorylation relative to the total ErbB-2 protein found in the cells. Once the phospho-ErbB2 / HER2 and total-ErbB2 / HER2 signals have been normalized for cell number, a comparison of the ratio of phosphorylated ErbB2 / HER2 to total ErbB2 / HER2 for each of the cell growth conditions can be made. The provided total-ErbB2 / HER2 antibody can be used as a positive control to demonstrate that the cells contain ErbB2 / HER2, the kit reagents are functional and that the protocol is performed correctly. Also, because fixed cells are stable for several weeks, many plates can be prepared simultaneously and then the assay performed when desired.
The ErbB2 / HER2proto-oncogene is comprised of an extracellular ligand binding domain, a signal transmembrane domain, a tyrosine kinase domain and a C terminal region that mediates signaling. ErbB-2 forms heterodimers with other members of the EGF receptor family, such as ErbB-1 (EGFR). ErbB2 / HER2 is autophosphorylated on five tyrosine residues at its C terminus, producing docking sites for SH2-domain containing proteins such as phospholipase Cγ, PI3-K, Src and CHK. These distinct tyrosine phosphorylation sites play an important role in the modulation of ErbB-2 mediated function.
Tyrosine 877 of ErbB2 / HER2 is homologous to pp60c-Src Tyr416 located in the kinase domain. Phosphorylation at this site is thought to be involved in regulation of ErbB-2 biological activity.
Tyrosine 1248 of ErbB2 / HER2 is one of the major auto-phosphorylation sites in this molecule. The phosphorylation of this site couples ErbB-2 to the Ras-Raf-MAP kinase signal transduction pathway.
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Storage instructionsPlease refer to protocols.
Components 1 x 96 tests 5 x 96 tests 1% SDS Solution 1 x 22ml 5 x 22ml 10% Triton X-100 1 x 10ml 5 x 10ml 10X PBS 1 x 120ml 5 x 120ml 1X Antibody Blocking Buffer 1 x 22ml 5 x 22ml 1X Antibody Dilution Buffer 1 x 30ml 5 x 30ml 96-well tissue culture plate 2 units 10 units Anti-rabbit HRP-conjugated IgG 4 x 11µl 20 x 11µl Crystal Violet Solution 1 x 22ml 5 x 22ml Developing Solution 2 x 11ml 10 x 11ml Phospho-ErB-2 (Y1248) antibody 1 x 9µl 5 x 9µl Plate sealer 2 units 10 units Stop Solution 2 x 11ml 10 x 11ml Total-ErbB-2 antibody 1 x 9µl 5 x 9µl
FunctionProtein tyrosine kinase that is part of several cell surface receptor complexes, but that apparently needs a coreceptor for ligand binding. Essential component of a neuregulin-receptor complex, although neuregulins do not interact with it alone. GP30 is a potential ligand for this receptor. Regulates outgrowth and stabilization of peripheral microtubules (MTs). Upon ERBB2 activation, the MEMO1-RHOA-DIAPH1 signaling pathway elicits the phosphorylation and thus the inhibition of GSK3B at cell membrane. This prevents the phosphorylation of APC and CLASP2, allowing its association with the cell membrane. In turn, membrane-bound APC allows the localization of MACF1 to the cell membrane, which is required for microtubule capture and stabilization.
In the nucleus is involved in transcriptional regulation. Associates with the 5'-TCAAATTC-3' sequence in the PTGS2/COX-2 promoter and activates its transcription. Implicated in transcriptional activation of CDKN1A; the function involves STAT3 and SRC. Involved in the transcription of rRNA genes by RNA Pol I and enhances protein synthesis and cell growth.
Tissue specificityExpressed in a variety of tumor tissues including primary breast tumors and tumors from small bowel, esophagus, kidney and mouth.
Involvement in diseaseHereditary diffuse gastric cancer
Chromosomal aberrations involving ERBB2 may be a cause gastric cancer. Deletions within 17q12 region producing fusion transcripts with CDK12, leading to CDK12-ERBB2 fusion leading to truncated CDK12 protein not in-frame with ERBB2.
Sequence similaritiesBelongs to the protein kinase superfamily. Tyr protein kinase family. EGF receptor subfamily.
Contains 1 protein kinase domain.
modificationsAutophosphorylated. Autophosphorylation occurs in trans, i.e. one subunit of the dimeric receptor phosphorylates tyrosine residues on the other subunit (Probable). Ligand-binding increases phosphorylation on tyrosine residues (PubMed:27134172). Signaling via SEMA4C promotes phosphorylation at Tyr-1248 (PubMed:17554007). Dephosphorylated by PTPN12 (PubMed:27134172).
Cellular localizationCytoplasm. Nucleus and Cell membrane. Cytoplasm, perinuclear region. Nucleus. Translocation to the nucleus requires endocytosis, probably endosomal sorting and is mediated by importin beta-1/KPNB1.
- Information by UniProt
- Verb b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog
- C erb B2/neu protein
Measurement of phospho-ErbB-2 (Y1248) and total ErbB2 / HER2. Human A431 cells were cultured in 96-well plates and serum-starved for 16 hours. Cells were then stimulated with 100 ng/ml of Epidermal Growth Factor for 10 minutes and fixed. Total ErbB-2 and ErbB-2 phosphorylated on Tyr1248 were each assayed using the phospho-ErbB-2 (Y1248) and total-ErbB-2 antibodies included in the kit. Data were plotted after correction for cell number (performed through use of Crystal Violet).
ab207465 has not yet been referenced specifically in any publications.