Rat Pro-neuregulin-1, membrane-bound isoform (Nrg1) ELISA Kit
The Rat Pro-Neuregulin-1 Membrane-Bound Isoform (NRG1) ELISA Kit is specifically designed for the accurate measurement of NRG1 levels in rat serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring accurate and reproducible results for a variety of research applications.NRG1 is a critical protein involved in various cellular processes, including neuronal development, synapse formation, and cardiac function.
Dysregulation of NRG1 has been linked to conditions such as schizophrenia, bipolar disorder, and cancer, highlighting its importance as a potential biomarker for disease research and therapeutic development.With its reliable performance and ease of use, the Rat Pro-Neuregulin-1 Membrane-Bound Isoform (NRG1) ELISA Kit is an essential tool for researchers looking to study NRG1 biology and its role in health and disease.
Product Name:
Rat Pro-neuregulin-1, membrane-bound isoform (Nrg1) ELISA Kit
SKU:
RTEB1243
Size:
96T
Target:
Rat Pro-neuregulin-1, membrane-bound isoform (Nrg1)
Synonyms:
Pro-NRG1, Ndf
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Rat
Detection Range:
0.78-50ng/mL
Sensitivity:
0.39ng/mL
Intra CV:
5.3%
Inter CV:
9.6%
Linearity:
Sample
1:2
1:4
1:8
1:16
Serum(N=5)
95-103%
98-108%
81-91%
100-108%
EDTA Plasma(N=5)
102-112%
105-115%
98-109%
97-109%
Heparin Plasma(N=5)
98-107%
109-119%
90-102%
84-96%
Recovery:
Sample Type
Average(%)
Recovery Range(%)
Serum
104
98-110
Plasma
106
100-112
Function:
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. Binds to ERBB4 and ERBB3. Acts as a ligand for integrins and binds (via EGF domain) to integrins ITGAV:ITGB3 or ITGA6:ITGB4. Its binding to integrins and subsequent ternary complex formation with integrins and ERRB3 are essential for NRG1-ERBB signaling (By similarity). Induces the phosphorylation and activation of MAPK3/ERK1, MAPK1/ERK2 and AKT1, and ligand-dependent ERBB4 endocytosis is essential for the NRG1-mediated activation of these kinases in neurons (PubMed:17250808).
Uniprot:
P43322
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant rat Pro-neuregulin-1, membrane-bound isoform
Sub Unit:
The cytoplasmic domain interacts with the LIM domain region of LIMK1 (PubMed:9685409). Forms a ternary complex with ERBB3 and ITGAV:ITGB3 or ITGA6:ITGB4.
Subcellular Location:
Neuregulin-1 Secreted
Storage:
Please see kit components below for exact storage details
Note:
For research use only
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: Ligand, receptor tyrosine kinase; Cell development/differentiation; Membrane protein, integral; Cytokine; Motility/polarity/chemotaxisCellular Component: extracellular space; integral to plasma membrane; axon; apical plasma membrane; cytoplasm; integral to membrane; plasma membrane; synapse; neuromuscular junctionMolecular Function: ErbB-2 class receptor binding; ErbB-3 class receptor binding; growth factor activity; transcription cofactor activity; protein tyrosine kinase activator activity; receptor bindingBiological Process: myelination; regulation of protein heterodimerization activity; muscle development; transmembrane receptor protein tyrosine kinase activation (dimerization); positive regulation of cell adhesion; wound healing; cellular protein complex disassembly; heart development; ventricular cardiac muscle cell differentiation; cell morphogenesis; locomotory behavior; glucose transport; synaptogenesis; glial cell differentiation; regulation of blood pressure; regulation of cell differentiation; organ development; cell differentiation; positive regulation of cardiac muscle cell proliferation; nervous system development; cell migration; neurotransmitter receptor metabolic process; MAPKKK cascade; neuron fate commitment; positive regulation of cell growth; peripheral nervous system development; cell proliferation; positive regulation of protein kinase B signaling cascade; glial cell fate commitment; positive regulation of protein kinase activity; 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
UniProt Protein Details:
NCBI Summary:
ligand for Erbb3 and Erbb4 receptors; gene produces many different alternative splicing isoforms; involved in neural and organ development [RGD, Feb 2006]
pro-neuregulin-1, membrane-bound isoform; pro-NRG1; neuregulin 1 type III beta 3
UniProt Protein Name:
Pro-neuregulin-1, membrane-bound isoform
UniProt Synonym Protein Names:
Acetylcholine receptor-inducing activity; ARIA; 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_RAT
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.
