Human D (2) dopamine receptor (DRD2) ELISA Kit

Product Name:	Human D (2)dopamine receptor (DRD2) ELISA Kit
SKU:	HUEB0794
Size:	96T
Target:	Human D (2)dopamine receptor (DRD2)
Synonyms:	Dopamine D2 receptor
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.312-20ng/mL
Sensitivity:	0.156ng/mL

Intra CV:

4.4%

Inter CV:

6.8%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	98-108%	105-114%	111-119%	84-84%
EDTA Plasma(N=5)	109-118%	103-112%	103-115%	111-120%
Heparin Plasma(N=5)	94-104%	101-111%	100-110%	113-122%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	87	81-93
Plasma	89	83-95

Function:

Dopamine receptor whose activity is mediated by G proteins which inhibit adenylyl cyclase.

Uniprot:	P14416
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human D(2) dopamine receptor
Sub Unit:	Forms homo- and heterooligomers with DRD4. The interaction with DRD4 may modulate agonist-induced downstream signaling. Interacts with GPRASP1, PPP1R9B and CLIC6 (By similarity). Interacts with CADPS and CADPS2. Interacts with ARRB2 (By similarity). Interacts with KCNA2 (By similarity). Interacts with GNAI2 isoform sGi2, the interaction allows the creation of an intracellular pool of DRD2 that can be released to cell surface upon agonist stimulation. Interacts with HTR2A.
Research Area:	Neurosciences
Subcellular Location:	Cell membrane Multi-pass membrane protein
Storage:	Please see kit components below for exact storage details
Note:	For research use only

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 Chromosomal Location of Human Ortholog: 11q23 Cellular Component: axon; dendrite; integral to plasma membrane; nonmotile primary cilium; plasma membrane; synaptic vesicle membrane Molecular Function:adrenoceptor activity; dopamine binding; dopamine D2 receptor-like receptor activity; drug binding; identical protein binding; protein binding Biological Process: adenohypophysis development; adult walking behavior; arachidonic acid secretion; associative learning; axonogenesis; behavioral response to cocaine; behavioral response to ethanol; branching morphogenesis of a nerve; cerebral cortex GABAergic interneuron migration; circadian regulation of gene expression; dopamine metabolic process; dopamine receptor, adenylate cyclase inhibiting pathway; dopamine receptor, phospholipase C activating pathway; locomotory behavior; negative regulation of blood pressure; negative regulation of cell migration; negative regulation of cell proliferation; negative regulation of dopamine receptor signaling pathway; negative regulation of protein kinase B signaling cascade; negative regulation of protein secretion; negative regulation of synaptic transmission, glutamatergic; nerve-nerve synaptic transmission; peristalsis; phosphatidylinositol metabolic process; positive regulation of cytokinesis; positive regulation of dopamine uptake; positive regulation of growth hormone secretion; positive regulation of neuroblast proliferation; prepulse inhibition; protein localization; reduction of cytosolic calcium ion concentration; regulation of cAMP metabolic process; regulation of dopamine secretion; regulation of dopamine uptake; regulation of heart rate; regulation of long-term neuronal synaptic plasticity; regulation of potassium ion transport; regulation of sodium ion transport; regulation of synaptic transmission, GABAergic; regulation of systemic arterial blood pressure by neurological process; release of sequestered calcium ion into cytosol; response to amphetamine; response to cocaine; response to drug; response to light stimulus; response to morphine; response to toxin; sensory perception of smell; synaptic transmission, dopaminergic; synaptogenesis; thermoregulation; visual learning Disease: Myoclonic Dystonia
NCBI Summary:	This gene encodes the D2 subtype of the dopamine receptor. This G-protein coupled receptor inhibits adenylyl cyclase activity. A missense mutation in this gene causes myoclonus dystonia; other mutations have been associated with schizophrenia. Alternative splicing of this gene results in two transcript variants encoding different isoforms. A third variant has been described, but it has not been determined whether this form is normal or due to aberrant splicing. [provided by RefSeq, Jul 2008]
UniProt Code:	P14416
NCBI GenInfo Identifier:	118206
NCBI Gene ID:	1813
NCBI Accession:	P14416.2
UniProt Secondary Accession:	P14416,Q9NZR3, Q9UPA9,
UniProt Related Accession:	P14416
Molecular Weight:	50,847 Da
NCBI Full Name:	D(2) dopamine receptor
NCBI Synonym Full Names:	dopamine receptor D2
NCBI Official Symbol:	DRD2
NCBI Official Synonym Symbols:	D2R; D2DR
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_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
Human Dopamine D2 R / DRD2 ELISA Kit	Anti-DRD2 Antibody (CAB12930)
Human D2R/DRD2 (Dopamine Receptor D2) CLIA Kit (HUES00698)	DRD1 Antibody (PACO08935)
Human D2R/DRD2 (Dopamine Receptor D2) ELISA Kit (HUES02292)