NRG1 Isoform-10 Colorimetric Cell-Based ELISA Kit (CBCAB00784)

Product Name:	NRG1 Isoform-10 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00784
ELISA Type:	Cell-Based
Target:	NRG1 isoform-10
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The NRG1 Isoform-10 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect NRG1 Isoform-10 protein expression profile in cells. The kit can be used for measuring the relative amounts of NRG1 Isoform-10 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on NRG1 Isoform-10.

Qualitative determination of NRG1 Isoform-10 concentration is achieved by an indirect ELISA format. In essence, NRG1 Isoform-10 is captured by NRG1 Isoform-10-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: 3084, UniProt ID: Q02297-10, OMIM: 142445, Unigene: Hs.453951
Gene Symbol:	NRG1
Sub Type:	None

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. 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
UniProt Protein Details:
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:	Q02297-10
NCBI GenInfo Identifier:	9297018
NCBI Gene ID:	3084
NCBI Accession:	Q02297.3
UniProt Secondary Accession:	Q02297-10,O14667, P98202, Q02298, Q02299, Q07110, Q07111 A5YAK4, A5YAK5, A8K1L2
UniProt Related Accession:	Q02297-10,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

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-NRG1 Isoform-10 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.)