Mouse D (2)dopamine receptor ELISA Kit (MOFI00274)

Product Name:	Mouse D (2)dopamine receptor ELISA Kit
Product Code:	MOFI00274
Size:	96 Assays
Alias:	Drd2, D2R, DRD2, Dopamine Receptor D2, Dopamine D2 receptor
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse Drd2 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.188ng/ml
Range:	0.313-20ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Mouse Drd2 and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse Drd2 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	86-103	90
EDTA plasma(n=5)	89-104	94
UFH plasma(n=5)	89-105	95

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse Drd2 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)	90-105%	97-105%	86-105%
EDTA plasma(n=5)	90-97%	83-101%	84-93%
UFH plasma(n=5)	81-97%	82-98%	82-95%

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

P61168

UniProt Protein Function:	DRD2: Dopamine receptor whose activity is mediated by G proteins which inhibit adenylyl cyclase. Defects in DRD2 are associated with dystonia type 11 (DYT11); also known as alcohol-responsive dystonia. DYT11 is a myoclonic dystonia. Dystonia is defined by the presence of sustained involuntary muscle contractions, often leading to abnormal postures. DYT11 is characterized by involuntary lightning jerks and dystonic movements and postures alleviated by alcohol. Inheritance is autosomal dominant. The age of onset, pattern of body involvement, presence of myoclonus and response to alcohol are all variable. Belongs to the G-protein coupled receptor 1 family. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Receptor, GPCR; GPCR, family 1; Membrane protein, multi-pass; Membrane protein, integral Cellular Component: synaptic vesicle membrane; integral to plasma membrane; dendrite; postsynaptic density; dendritic spine; integral to membrane; acrosome; perikaryon; cytosol; membrane; endocytic vesicle; axon; plasma membrane; cytoplasmic vesicle; nerve terminal; lateral plasma membrane Molecular Function:G-protein coupled receptor activity; dopamine D2 receptor-like receptor activity; identical protein binding; ionotropic glutamate receptor binding; signal transducer activity; protein homodimerization activity; dopamine receptor activity; protein heterodimerization activity; dopamine binding; drug binding; receptor binding Biological Process: elevation of cytosolic calcium ion concentration during G-protein signaling, coupled to IP3 second messenger (phospholipase C activating); positive regulation of dopamine uptake; prepulse inhibition; positive regulation of receptor internalization; thermoregulation; positive regulation of multicellular organism growth; regulation of phosphoprotein phosphatase activity; adult walking behavior; dopamine metabolic process; negative regulation of insulin secretion; dopamine receptor, adenylate cyclase inhibiting pathway; protein localization; negative regulation of blood pressure; phosphatidylinositol metabolic process; response to drug; positive regulation of neuroblast proliferation; response to light stimulus; cerebral cortex GABAergic interneuron migration; negative regulation of synaptic transmission, glutamatergic; regulation of sodium ion transport; arachidonic acid secretion; regulation of synaptic transmission, GABAergic; G-protein signaling, coupled to cAMP nucleotide second messenger; positive regulation of growth hormone secretion; acid secretion; G-protein coupled receptor internalization; reduction of cytosolic calcium ion concentration; regulation of MAPKKK cascade; activation of protein kinase activity; dopamine receptor signaling pathway; positive regulation of transcription from RNA polymerase II promoter; regulation of long-term neuronal synaptic plasticity; synaptic transmission, dopaminergic; peristalsis; startle response; branching morphogenesis of a nerve; negative regulation of circadian sleep/wake cycle, sleep; response to morphine; locomotory behavior; behavioral response to ethanol; signal transduction; regulation of dopamine secretion; negative regulation of cell proliferation; synaptic transmission; behavioral response to cocaine; forebrain development; adenohypophysis development; visual learning; nerve-nerve synaptic transmission; feeding behavior; circadian regulation of gene expression; negative regulation of dopamine secretion; negative regulation of cell migration; associative learning; grooming behavior; regulation of synaptic transmission; positive regulation of cytokinesis; negative regulation of protein secretion; Wnt receptor signaling pathway; negative regulation of protein kinase B signaling cascade; regulation of heart rate; regulation of potassium ion transport; regulation of cAMP metabolic process; sensory perception of smell; negative regulation of adenylate cyclase activity; response to amphetamine; regulation of dopamine uptake; auditory behavior; response to cocaine; G-protein coupled receptor protein signaling pathway; regulation of systemic arterial blood pressure by neurological process; negative regulation of dopamine receptor signaling pathway; axonogenesis; pigmentation; long-term memory; positive regulation of G-protein coupled receptor protein signaling pathway; release of sequestered calcium ion into cytosol; adult behavior; response to hypoxia; regulation of synapse structural plasticity; dopamine receptor, phospholipase C activating pathway
UniProt Code:	P61168
NCBI GenInfo Identifier:	148747212
NCBI Gene ID:	13489
NCBI Accession:	NP_034207.2
UniProt Secondary Accession:	P61168,P13953, Q0VGH9,
UniProt Related Accession:	P61168
Molecular Weight:	47,625 Da
NCBI Full Name:	D(2) dopamine receptor
NCBI Synonym Full Names:	dopamine receptor D2
NCBI Official Symbol:	Drd2
NCBI Official Synonym Symbols:	D2R; Drd-2
NCBI Protein Information:	D(2) dopamine receptor
UniProt Protein Name:	D(2) dopamine receptor
UniProt Synonym Protein Names:	Dopamine D2 receptor
Protein Family:	D(2) dopamine receptor
UniProt Gene Name:	Drd2
UniProt Entry Name:	DRD2_MOUSE

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

Step	Procedure
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.

Mouse D (2)dopamine receptor ELISA Kit (MOFI00274)

Description