Human OPRM1 ELISA Kit (HUFI00776)- High Sensitivity

Product Name:	Human OPRM1 (Mu-type opioid receptor) ELISA Kit
Product Code:	HUFI00776
Size:	96 Assays
Alias:	OPRM1, Mu-type opioid receptor, LMOR, MOP, MOR, MOR1, hMOP, M-OR-1, MOR-1, Mu opiate receptor, opioid receptor, mu 1, OPRM
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human OPRM1 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 OPRM1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human OPRM1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-101	94
EDTA plasma(n=5)	85-101	93
UFH plasma(n=5)	86-105	96

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human OPRM1 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)	87-102%	88-103%	89-101%
EDTA plasma(n=5)	83-101%	84-100%	86-98%
UFH plasma(n=5)	81-98%	80-97%	88-96%

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

P35372

UniProt Protein Function:	MOR-1: a Gi-protein-coupled receptor for beta-endorphin, morphine and other opiates. Inhibits neurotransmitter release by reducing calcium ion currents and increasing potassium ion conductance. Ligand-binding inactivates adenylyl cyclase, and activates a variety of G-beta-gamma-dependent pathways including the MAPK and the PI3K/Akt cascades. Two splice-variant isoforms have been described.
UniProt Protein Details:	Protein type:GPCR, family 1; Membrane protein, multi-pass; Receptor, GPCR; Membrane protein, integral Chromosomal Location of Human Ortholog: 6q24-q25 Cellular Component: dendrite cytoplasm; Golgi apparatus; neuron projection; focal adhesion; endoplasmic reticulum; integral to plasma membrane; plasma membrane; perikaryon; sarcolemma; lipid raft Molecular Function:G-protein coupled receptor activity; voltage-gated calcium channel activity; neuropeptide binding; protein domain specific binding; protein binding; G-protein alpha-subunit binding; G-protein beta-subunit binding; filamin binding; beta-endorphin receptor activity Biological Process: response to food; positive regulation of neurogenesis; positive regulation of nitric oxide biosynthetic process; wound healing; negative regulation of nitric oxide biosynthetic process; cellular response to stress; negative regulation of adenylate cyclase activity; locomotory behavior; response to lipopolysaccharide; behavioral response to ethanol; sensory perception of pain; response to cocaine; G-protein signaling, coupled to cyclic nucleotide second messenger; negative regulation of cell proliferation; synaptic transmission; elevation of cytosolic calcium ion concentration; neuropeptide signaling pathway; reduction of cytosolic calcium ion concentration; positive regulation of appetite; sensory perception; opioid receptor, adenylate cyclase inhibiting pathway; G-protein signaling, coupled to IP3 second messenger (phospholipase C activating); regulation of sensory perception of pain; regulation of excitatory postsynaptic membrane potential; dopamine receptor, adenylate cyclase activating pathway; acute inflammatory response to antigenic stimulus Disease: Epilepsy, Idiopathic Generalized
NCBI Summary:	This gene encodes one of at least three opioid receptors in humans; the mu opioid receptor (MOR). The MOR is the principal target of endogenous opioid peptides and opioid analgesic agents such as beta-endorphin and enkephalins. The MOR also has an important role in dependence to other drugs of abuse, such as nicotine, cocaine, and alcohol via its modulation of the dopamine system. The NM_001008503.2:c.118A>G allele has been associated with opioid and alcohol addiction and variations in pain sensitivity but evidence for it having a causal role is conflicting. Multiple transcript variants encoding different isoforms have been found for this gene. Though the canonical MOR belongs to the superfamily of 7-transmembrane-spanning G-protein-coupled receptors some isoforms of this gene have only 6 transmembrane domains. [provided by RefSeq, Oct 2013]
UniProt Code:	P35372
NCBI GenInfo Identifier:	2851402
NCBI Gene ID:	4988
NCBI Accession:	P35372.2
UniProt Secondary Accession:	P35372,Q12930, Q4VWM1, Q4VWM2, B0FXJ1, B2R9S7, B8Q1L7 B8Q1L8, B8Q1L9, E7EWZ3, G8XRH6, G8XRH8,
UniProt Related Accession:	P35372
Molecular Weight:	20,188 Da
NCBI Full Name:	Mu-type opioid receptor
NCBI Synonym Full Names:	opioid receptor, mu 1
NCBI Official Symbol:	OPRM1
NCBI Official Synonym Symbols:	MOP; MOR; LMOR; MOR1; OPRM; M-OR-1
NCBI Protein Information:	mu-type opioid receptor; mu opiate receptor; mu opioid receptor hMOR-1a
UniProt Protein Name:	Mu-type opioid receptor
UniProt Synonym Protein Names:	Mu opiate receptor; Mu opioid receptor; MOP; hMOP
Protein Family:	Mu-type opioid receptor
UniProt Gene Name:	OPRM1
UniProt Entry Name:	OPRM_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 µl 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 µl 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.