Human ERBB2 ELISA Kit (HUEB0292)- High Sensitivity

Product Name:	Human Receptor tyrosine-protein kinase erbB-2 (ERBB2) ELISA Kit
SKU:	HUEB0292
Size:	96T
Target:	Human Receptor tyrosine-protein kinase erbB-2 (ERBB2)
Synonyms:	Metastatic lymph node gene 19 protein, Proto-oncogene Neu, Proto-oncogene c-ErbB-2, Tyrosine kinase-type cell surface receptor HER2, p185erbB2, MLN 19, CD340, HER2, MLN19, NEU, NGL
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.312-20ng/mL
Sensitivity:	0.1ng/mL

Intra CV:

5.6%

Inter CV:

10.2%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	86-95%	90-99%	95-106%	107-117%
EDTA Plasma(N=5)	111-120%	105-115%	87-97%	84-95%
Heparin Plasma(N=5)	99-109%	88-97%	80-90%	106-118%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	107	101-113
Plasma	109	103-115

Function:

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.

Uniprot:	P04626
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Receptor tyrosine-protein kinase erbB-2
Sub Unit:	Homodimer (PubMed:21454582). Heterodimer with EGFR, ERBB3 and ERBB4 (PubMed:10358079, PubMed:15093539, PubMed:21190959, PubMed:16978839). Part of a complex with EGFR and either PIK3C2A or PIK3C2B. May interact with PIK3C2B when phosphorylated on Tyr-1196 (PubMed:10805725). Interacts with PLXNB1 (PubMed:15210733). Interacts (when phosphorylated on Tyr-1248) with MEMO1 (PubMed:15156151). Interacts with MUC1; the interaction is enhanced by heregulin (HRG) (PubMed:12939402). Interacts (when phosphorylated on Tyr-1139) with GRB7 (via SH2 domain) (PubMed:12975581). Interacts (when phosphorylated on Tyr-1248) with ERBIN (PubMed:12444095). Interacts with KPNB1, RANBP2, EEA1, CRM1 and CLTC (PubMed:16314522). Interacts with PTK6 (PubMed:18719096). Interacts with RPA194 and ACTB (PubMed:21555369). Interacts with PRKCABP, SRC and MYOC (By similarity). Interacts (preferentially with the tyrosine phosphorylated form) with CPNE3; this interaction occurs at the cell membrane and is increased in a growth factor heregulin-dependent manner (PubMed:20010870). Interacts with HSP90AA1 and HSP90AB1 in an ATP-dependent manner; the interaction suppresses ERBB2 kinase activity (PubMed:26517842).
Research Area:	Cancer
Subcellular Location:	Isoform 3 Cytoplasm Nucleus
Storage:	Please see kit components below for exact storage details
Note:	For research use only

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 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

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

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

ELISA	Antibodies
HER2 (Phospho-Tyr1221/Tyr1222) Colorimetric Cell-Based ELISA Kit	Anti-HER2 / ErbB2 Antibody (CAB2071)[KO Validated]
HER2 Colorimetric Cell-Based ELISA Kit	Anti-Phospho-ERBB2-Y1221/Y1222 Antibody (CABP0149)
HER2 (Phospho-Tyr1248) Colorimetric Cell-Based ELISA Kit	Anti-Phospho-ERBB2-Y1221/1222 Antibody (CABP0527)
	Anti-Phospho-ERBB2-Y1248 Antibody (CABP0581)
	Anti-CD68 Antibody (MACO0024)