Human GRIN1 ELISA Kit (HUEB0596)- High Sensitivity

Product Name:	Human Glutamate [NMDA] receptor subunit zeta-1 (GRIN1) ELISA Kit
SKU:	HUEB0596
Size:	96T
Target:	Human Glutamate [NMDA] receptor subunit zeta-1 (GRIN1)
Synonyms:	Glutamate [NMDA] receptor subunit zeta-1, N-methyl-D-aspartate receptor subunit NR1, NMD-R1, GluN1, NMDAR1
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.156-10ng/mL
Sensitivity:	0.078ng/mL

Intra CV:

4.7%

Inter CV:

9.8%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	88-97%	109-117%	94-106%	104-114%
EDTA Plasma(N=5)	95-103%	85-98%	97-107%	111-111%
Heparin Plasma(N=5)	81-94%	105-114%	93-103%	98-107%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	88	82-94
Plasma	90	84-96

Function:

Component of NMDA receptor complexes that function as heterotetrameric, ligand-gated ion channels with high calcium permeability and voltage-dependent sensitivity to magnesium. Channel activation requires binding of the neurotransmitter glutamate to the epsilon subunit, glycine binding to the zeta subunit, plus membrane depolarization to eliminate channel inhibition by Mg(2+) (PubMed:7685113, PubMed:28126851, PubMed:26919761, PubMed:26875626, PubMed:28105280). Sensitivity to glutamate and channel kinetics depend on the subunit composition (PubMed:26919761).

Uniprot:	Q05586
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Glutamate receptor ionotropic, NMDA 1
Sub Unit:	Heterotetramer. Forms heterotetrameric channels composed of two zeta subunits (GRIN1), and two epsilon subunits (GRIN2A, GRIN2B, GRIN2C or GRIN2D) (in vitro) (PubMed:7685113, PubMed:28126851, PubMed:26919761, PubMed:26875626, PubMed:28105280). Can also form heterotetrameric channels that contain at least one zeta subunit (GRIN1), an epsilon subunit, plus GRIN3A or GRIN3B (in vitro). In vivo, the subunit composition may vary in function of the expression levels of the different subunits. Found in a complex with GRIN2A or GRIN2B, GRIN3A and PPP2CB (By similarity). Found in a complex with GRIN2A or GRIN2B and GRIN3B (By similarity). Interacts with SNX27 (via PDZ domain); the interaction is required for recycling to the plasma membrane when endocytosed and prevent degradation in lysosomes (By similarity). Interacts with DLG4 and MPDZ. Interacts with LRFN1 and LRFN2 (By similarity). Interacts with MYZAP (PubMed:18849881). Found in a complex with DLG4 and PRR7 (By similarity). Found in a complex with GRIN2B and PRR7 (PubMed:27458189). Interacts with PRR7; the interaction is reduced following NMDA receptor activity (PubMed:27458189).
Research Area:	Neurosciences
Subcellular Location:	Cell membrane Multi-pass membrane protein Cell junction Synapse Postsynaptic cell membrane Cell junction Synapse Postsynaptic cell membrane Postsynaptic density Enriched in postsynaptic plasma membrane and postsynaptic densities.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	NMDAR1: a subunit of N-methyl-D-aspartate (NMDA) receptors, members of the glutamate receptor channel superfamily. Possesses high calcium permeability and voltage-dependent sensitivity to magnesium and is modulated by glycine. Plays a key role in synaptic plasticity, synaptogenesis, excitotoxicity, memory acquisition and learning. Mediates neuronal functions in glutamate neurotransmission. Three alternatively-spliced isoforms have been described.
UniProt Protein Details:	Protein type:Channel, ligand-gated; Membrane protein, multi-pass; Membrane protein, integral; Channel, calcium Chromosomal Location of Human Ortholog: 9q34.3 Cellular Component: neuron projection; cell surface; endoplasmic reticulum; integral to plasma membrane; postsynaptic density; dendrite; dendritic spine; terminal button; excitatory synapse; N-methyl-D-aspartate selective glutamate receptor complex; postsynaptic membrane; synaptic vesicle; plasma membrane; synapse; cell junction Molecular Function:voltage-gated cation channel activity; neurotransmitter binding; glutamate receptor binding; calcium channel activity; calcium ion binding; calmodulin binding; protein binding; enzyme binding; glutamate binding; extracellular-glutamate-gated ion channel activity; protein heterodimerization activity; N-methyl-D-aspartate selective glutamate receptor activity; glycine binding Biological Process: regulation of long-term neuronal synaptic plasticity; axon guidance; male mating behavior; prepulse inhibition; adult locomotory behavior; positive regulation of apoptosis; regulation of dendrite morphogenesis; rhythmic process; response to morphine; sensory perception of pain; regulation of axonogenesis; calcium ion homeostasis; synaptic transmission; regulation of respiratory gaseous exchange; conditioned taste aversion; ephrin receptor signaling pathway; visual learning; negative regulation of neuron apoptosis; protein tetramerization; cation transport; synaptic transmission, glutamatergic; response to amphetamine; social behavior; respiratory gaseous exchange; pons maturation; cellular calcium ion homeostasis; regulation of membrane potential; response to ethanol; regulation of synaptogenesis; olfactory learning; long-term memory; suckling behavior; propylene metabolic process; ionotropic glutamate receptor signaling pathway; positive regulation of transcription from RNA polymerase II promoter; cerebral cortex development; regulation of excitatory postsynaptic membrane potential; response to calcium ion Disease: Mental Retardation, Autosomal Dominant 8
NCBI Summary:	The protein encoded by this gene is a critical subunit of N-methyl-D-aspartate receptors, members of the glutamate receptor channel superfamily which are heteromeric protein complexes with multiple subunits arranged to form a ligand-gated ion channel. These subunits play a key role in the plasticity of synapses, which is believed to underlie memory and learning. Cell-specific factors are thought to control expression of different isoforms, possibly contributing to the functional diversity of the subunits. Alternatively spliced transcript variants have been described. [provided by RefSeq, Jul 2008]
UniProt Code:	Q05586
NCBI GenInfo Identifier:	548377
NCBI Gene ID:	2902
NCBI Accession:	Q05586.1
UniProt Secondary Accession:	Q05586,P35437, Q12867, Q12868, Q5VSF3, Q5VSF4, Q5VSF5 Q5VSF6, Q5VSF7, A6NLK7, A6NLR1, C9K0X1,
UniProt Related Accession:	Q05586
Molecular Weight:	101,851 Da
NCBI Full Name:	Glutamate receptor ionotropic, NMDA 1
NCBI Synonym Full Names:	glutamate receptor, ionotropic, N-methyl D-aspartate 1
NCBI Official Symbol:	GRIN1
NCBI Official Synonym Symbols:	NR1; MRD8; GluN1; NMDA1; NMDAR1
NCBI Protein Information:	glutamate receptor ionotropic, NMDA 1; NMD-R1; glutamate [NMDA] receptor subunit zeta 1; glutamate [NMDA] receptor subunit zeta-1; N-methyl-D-aspartate receptor subunit NR1; N-methyl-D-aspartate receptor channel, subunit zeta-1
UniProt Protein Name:	Glutamate receptor ionotropic, NMDA 1
UniProt Synonym Protein Names:	Glutamate [NMDA] receptor subunit zeta-1; N-methyl-D-aspartate receptor subunit NR1; NMD-R1
Protein Family:	Glutamate receptor ionotropic
UniProt Gene Name:	GRIN1
UniProt Entry Name:	NMDZ1_HUMAN

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
NMDAR1 Colorimetric Cell-Based ELISA Kit	Anti-NMDAR1 Antibody (CAB11699)
NMDAR1 (Phospho-Ser896) Colorimetric Cell-Based ELISA Kit	Anti-GRIN1 Antibody (CAB13995)
NMDAR1 (Phospho-Ser897) Colorimetric Cell-Based ELISA Kit	Anti-GRIN1 Antibody (CAB7167)
	Anti-GRIN1 Antibody (CAB7677)
	Anti-Phospho-GRIN1-S896 Antibody (CABP0165)