HER2 (Phospho-Tyr1248)Colorimetric Cell-Based ELISA Kit (CBCAB00469)

Product Name:	HER2 (Phospho-Tyr1248)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00469
ELISA Type:	Cell-Based
Target:	HER2 (Phospho-Tyr1248)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The HER2 (Phospho-Tyr1248) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect HER2 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated HER2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on HER2 phosphorylation.

Qualitative determination of HER2 (Phospho-Tyr1248) concentration is achieved by an indirect ELISA format. In essence, HER2 (Phospho-Tyr1248) is captured by HER2 (Phospho-Tyr1248)-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1.	A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2.	Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.

Database Information:	Gene ID: 2064, UniProt ID: P04626, OMIM: 164870, Unigene: Hs.446352
Gene Symbol:	ERBB2
Sub Type:	Phospho

UniProt Protein Function:	HER2: a proto-oncogenic receptor tyrosine kinase of the EGFR family. Essential component of a neuregulin-receptor complex, although neuregulins do not interact with it alone. Not activated by EGF, TGF- alpha and amphiregulin. Amplified in breast cancer. Overexpression induces constitutive activity, and the gene is amplified or overexpressed in up to 30% of breast cancers, correlating with poor survival. The antibody Herceptin is approved for treatment of metastatic breast cancer with HER2 amplification/overexpression. Somatic mutations seen in 4% of lung cancers and also in breast, gastric, ovarian cancer and glioblastoma. One SNP shows predisposition to breast and gastric cancer. Inhibitors: Herceptin, lapatinib, PKI-166, EKB-569, CI-1033.
UniProt Protein Details:	Protein type:EC 2.7.10.1; EGFR family; Kinase, protein; Membrane protein, integral; Oncoprotein; Protein kinase, TK; Protein kinase, tyrosine (receptor); TK group Chromosomal Location of Human Ortholog: 17q12 Cellular Component: basolateral plasma membrane; cytoplasm; endosome membrane; nucleus; plasma membrane; receptor complex Molecular Function:ErbB-3 class receptor binding; growth factor binding; identical protein binding; phosphatidylinositol-4,5-bisphosphate 3-kinase activity; protein binding; protein C-terminus binding; protein heterodimerization activity; protein phosphatase binding; protein-tyrosine kinase activity; Ras guanyl-nucleotide exchange factor activity; transmembrane receptor activity; transmembrane receptor protein tyrosine kinase activity Biological Process: cell proliferation; cell surface receptor linked signal transduction; enzyme linked receptor protein signaling pathway; MAPKKK cascade; phosphoinositide 3-kinase cascade; phosphoinositide-mediated signaling; positive regulation of cell adhesion; positive regulation of cell growth; positive regulation of epithelial cell proliferation; positive regulation of GTPase activity; positive regulation of MAP kinase activity; positive regulation of protein amino acid phosphorylation; positive regulation of transcription from RNA polymerase I promoter; positive regulation of transcription from RNA polymerase III promoter; positive regulation of translation; protein amino acid autophosphorylation; protein amino acid phosphorylation; regulation of microtubule-based process; regulation of phosphoinositide 3-kinase cascade; signal transduction; transmembrane receptor protein tyrosine kinase signaling pathway; wound healing Disease: Gastric Cancer; Glioma Susceptibility 1; Lung Cancer
NCBI Summary:	This gene encodes a member of the epidermal growth factor (EGF) receptor family of receptor tyrosine kinases. This protein has no ligand binding domain of its own and therefore cannot bind growth factors. However, it does bind tightly to other ligand-bound EGF receptor family members to form a heterodimer, stabilizing ligand binding and enhancing kinase-mediated activation of downstream signalling pathways, such as those involving mitogen-activated protein kinase and phosphatidylinositol-3 kinase. Allelic variations at amino acid positions 654 and 655 of isoform a (positions 624 and 625 of isoform b) have been reported, with the most common allele, Ile654/Ile655, shown here. Amplification and/or overexpression of this gene has been reported in numerous cancers, including breast and ovarian tumors. Alternative splicing results in several additional transcript variants, some encoding different isoforms and others that have not been fully characterized. [provided by RefSeq, Jul 2008]
UniProt Code:	P04626
NCBI GenInfo Identifier:	119533
NCBI Gene ID:	2064
NCBI Accession:	P04626.1
UniProt Secondary Accession:	P04626,Q14256, Q6LDV1, Q9UMK4, B2RZG3, B4DHN3, X5D2V5
UniProt Related Accession:	P04626
Molecular Weight:	97,382 Da
NCBI Full Name:	Receptor tyrosine-protein kinase erbB-2
NCBI Synonym Full Names:	erb-b2 receptor tyrosine kinase 2
NCBI Official Symbol:	ERBB2
NCBI Official Synonym Symbols:	NEU; NGL; HER2; TKR1; CD340; HER-2; MLN 19; HER-2/neu
NCBI Protein Information:	receptor tyrosine-protein kinase erbB-2
UniProt Protein Name:	Receptor tyrosine-protein kinase erbB-2
UniProt Synonym Protein Names:	Metastatic lymph node gene 19 protein; MLN 19; Proto-oncogene Neu; Proto-oncogene c-ErbB-2; Tyrosine kinase-type cell surface receptor HER2; p185erbB2; CD_antigen: CD340
UniProt Gene Name:	ERBB2

Component	Quantity
96-Well Cell Culture Clear-Bottom Microplate	2 plates
10X TBS	24 mL
Quenching Buffer	24 mL
Blocking Buffer	50 mL
15X Wash Buffer	50 mL
Primary Antibody Diluent	12 mL
100x Anti-Phospho Target Antibody	60 µL
100x Anti-Target Antibody	60 µL
Anti-GAPDH Antibody	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
Graph paper or computer software capable of generating or displaying logarithmic functions
Absorbent papers or vacuum aspirator
Test tubes or microfuge tubes capable of storing ≥1 ml
Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
Orbital shaker (optional)
Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1.	Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.	Incubate the cells for overnight at 37°C, 5% CO2.
3.	Treat the cells as desired.
4.	Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.	Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.	Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week.
7.	Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.	Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.	Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.	Add 50 µL of 1x primary antibodies Anti-HER2 (Phospho-Tyr1248) Antibody, Anti-HER2 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.	Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.	Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.	Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)