Human Neuregulin 1/NRG1 ELISA Kit (HUFI01655)

Product Name:	Human Neuregulin 1/NRG1 ELISA Kit
Product Code:	HUFI01655
Size:	96 Assays
Alias:	NRG-1, Neuregulin 1, Heregulin-1, HGL, HRG1-alpha, HRG1-beta 1, NRG1, ARIA, GGF2, GGF, glial growth factor, HGLneu differentiation factor, HRG, HRG1, HRGA, neuregulin 1 type IV beta 1a, MST131, MSTP131, NDFheregulin, alpha, 45kD, ERBB2 p185-activator, neuregulin 1, neuregulin 1 type IV beta 3, pro-neuregulin-1, membrane-bound isoform, pro-NRG1, sensory and motor neuron derived factor, SMDF
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human NRG-1 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human NRG-1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human NRG-1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	92-105	99
EDTA plasma(n=5)	87-99	93
UFH plasma(n=5)	85-105	96

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human NRG-1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	86-105%	85-100%	88-97%
EDTA plasma(n=5)	82-101%	83-100%	85-93%
UFH plasma(n=5)	85-99%	83-95%	83-97%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	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

Uniprot

Q02297

UniProt Protein Function:	NRG1: Direct ligand for ERBB3 and ERBB4 tyrosine kinase receptors. Concomitantly recruits ERBB1 and ERBB2 coreceptors, resulting in ligand-stimulated tyrosine phosphorylation and activation of the ERBB receptors. The multiple isoforms perform diverse functions such as inducing growth and differentiation of epithelial, glial, neuronal, and skeletal muscle cells; inducing expression of acetylcholine receptor in synaptic vesicles during the formation of the neuromuscular junction; stimulating lobuloalveolar budding and milk production in the mammary gland and inducing differentiation of mammary tumor cells; stimulating Schwann cell proliferation; implication in the development of the myocardium such as trabeculation of the developing heart. Isoform 10 may play a role in motor and sensory neuron development. The cytoplasmic domain interacts with the LIM domain region of LIMK1. Interacts with ERBB3 and ERBB4. Type I isoforms are the predominant forms expressed in the endocardium. Isoform alpha is expressed in breast, ovary, testis, prostate, heart, skeletal muscle, lung, placenta liver, kidney, salivary gland, small intestine and brain, but not in uterus, stomach, pancreas, and spleen. Isoform 3 is the predominant form in mesenchymal cells and in non-neuronal organs, whereas isoform 6 is the major neuronal form. Isoform 8 is expressed in spinal cord and brain. Isoform 9 is the major form in skeletal muscle cells; in the nervous system it is expressed in spinal cord and brain. Also detected in adult heart, placenta, lung, liver, kidney, and pancreas. Isoform 10 is expressed in nervous system: spinal cord motor neurons, dorsal root ganglion neurons, and brain. Predominant isoform expressed in sensory and motor neurons. Not detected in adult heart, placenta, lung, liver, skeletal muscle, kidney, and pancreas. Not expressed in fetal lung, liver and kidney. Type IV isoforms are brain-specific. Belongs to the neuregulin family. 10 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Cell development/differentiation; Membrane protein, integral; Ligand, receptor tyrosine kinase; Cytokine; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 8p12 Cellular Component: extracellular space; membrane; axon; integral to plasma membrane; apical plasma membrane; cytoplasm; extracellular region; neuromuscular junction; nucleus Molecular Function:ErbB-2 class receptor binding; protein binding; transmembrane receptor protein tyrosine kinase activator activity; growth factor activity; ErbB-3 class receptor binding; cytokine activity; transcription cofactor activity; protein tyrosine kinase activator activity; receptor tyrosine kinase binding; receptor binding Biological Process: regulation of protein heterodimerization activity; transmembrane receptor protein tyrosine kinase activation (dimerization); positive regulation of cell adhesion; neural crest cell development; wound healing; nerve growth factor receptor signaling pathway; cellular protein complex disassembly; cell morphogenesis; ventricular cardiac muscle cell differentiation; locomotory behavior; positive regulation of striated muscle cell differentiation; cardiac muscle cell differentiation; synaptogenesis; mammary gland development; cell communication; positive regulation of cardiac muscle cell proliferation; epidermal growth factor receptor signaling pathway; nervous system development; cell migration; phosphoinositide-mediated signaling; fibroblast growth factor receptor signaling pathway; neurotransmitter receptor metabolic process; regulation of protein homodimerization activity; MAPKKK cascade; neuron fate commitment; positive regulation of cell growth; peripheral nervous system development; positive regulation of protein kinase B signaling cascade; cell proliferation; embryonic development; glial cell fate commitment; innate immune response; negative regulation of secretion; positive regulation of Ras protein signal transduction; negative regulation of protein catabolic process; negative regulation of transcription, DNA-dependent; transmembrane receptor protein tyrosine kinase signaling pathway Disease: Schizophrenia 6
NCBI Summary:	The protein encoded by this gene is a membrane glycoprotein that that mediates cell-cell signaling and plays a critical role in the growth and development of multiple organ systems. An extraordinary variety of different isoforms are produced from this gene through alternative promoter usage and splicing. These isoforms are expressed in a tissue-specific manner and differ significantly in their structure, and are classified as types I, II, III, IV, V and VI. Dysregulation of this gene has been linked to diseases such as cancer, schizophrenia, and bipolar disorder (BPD). [provided by RefSeq, Jun 2014]
UniProt Code:	Q02297
NCBI GenInfo Identifier:	9297018
NCBI Gene ID:	3084
NCBI Accession:	Q02297.3
UniProt Secondary Accession:	Q02297,O14667, P98202, Q02298, Q02299, Q07110, Q07111 A5YAK4, A5YAK5, A8K1L2, B7Z4Z3, E9PHH4,
UniProt Related Accession:	Q02297
Molecular Weight:
NCBI Full Name:	Pro-neuregulin-1, membrane-bound isoform
NCBI Synonym Full Names:	neuregulin 1
NCBI Official Symbol:	NRG1
NCBI Official Synonym Symbols:	GGF; HGL; HRG; NDF; ARIA; GGF2; HRG1; HRGA; SMDF; MST131; MSTP131; NRG1-IT2
NCBI Protein Information:	pro-neuregulin-1, membrane-bound isoform; pro-NRG1; glial growth factor; neu differentiation factor; sensory and motor neuron derived factor; heregulin, alpha (45kD, ERBB2 p185-activator)
UniProt Protein Name:	Pro-neuregulin-1, membrane-bound isoform
UniProt Synonym Protein Names:	Acetylcholine receptor-inducing activity; ARIA; Breast cancer cell differentiation factor p45; Glial growth factor; Heregulin; HRG; Neu differentiation factor; Sensory and motor neuron-derived factor
Protein Family:	Pro-neuregulin
UniProt Gene Name:	NRG1
UniProt Entry Name:	NRG1_HUMAN

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

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 Âµl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 Âµl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

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.