Rat DRD1/Dopamine Receptor D1 ELISA Kit (RTFI00358)

Product Name:	Rat DRD1/Dopamine Receptor D1 ELISA Kit
Product Code:	RTFI00358
Size:	96 Assays
Target:	Rat Drd1
Alias:	Drd1, D1R, DRD1, Dopamine Receptor D1, Dopamine D1 receptor
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
Sensitivity:	7.5ng/ml
Range:	12.5-800ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Rat Drd1 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat Drd1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	90-101	97
EDTA plasma(n=5)	85-97	93
UFH plasma(n=5)	90-105	99

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat Drd1 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)	96-105%	85-102%	86-103%
EDTA plasma(n=5)	84-99%	83-91%	85-96%
UFH plasma(n=5)	81-94%	87-100%	88-100%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

P18901

UniProt Protein Function:	DRD1: a G-protein coupled receptor.One of the five types (D1 to D5) of receptors for dopamine. The most abundant dopamine receptor in the central nervous system. The activity of this receptor is mediated by G proteins which activate adenylyl cyclase. Interacts with calcyon.
UniProt Protein Details:	Protein type:Receptor, GPCR; Membrane protein, multi-pass; Membrane protein, integral; GPCR, family 1 Cellular Component: axon; caveola; cell soma; cytosol; dendrite; dendritic shaft; dendritic spine; endomembrane system; endoplasmic reticulum; endoplasmic reticulum membrane; integral to membrane; integral to plasma membrane; membrane; nerve terminal; nonmotile primary cilium; nucleus; plasma membrane Molecular Function:angiotensin receptor binding; ATPase binding; D3 dopamine receptor binding; dopamine binding; dopamine D1 receptor-like receptor activity; dopamine receptor activity; drug binding; G-protein alpha-subunit binding; G-protein coupled receptor activity; protein binding; protein complex binding; protein heterodimerization activity; protein phosphatase binding; receptor binding Biological Process: adenylate cyclase activation; adult walking behavior; associative learning; astrocyte development; behavioral fear response; behavioral response to cocaine; calcium-mediated signaling; cellular response to insulin stimulus; cerebral cortex GABAergic interneuron migration; conditioned taste aversion; dentate gyrus development; dopamine receptor signaling pathway; dopamine receptor, adenylate cyclase activating pathway; dopamine receptor, phospholipase C activating pathway; dopamine transport; elevation of cytosolic calcium ion concentration during G-protein signaling, coupled to IP3 second messenger (phospholipase C activating); feeding behavior; G-protein coupled receptor protein signaling pathway; G-protein signaling, adenylate cyclase activating pathway; G-protein signaling, coupled to cyclic nucleotide second messenger; generation of action potential; glucose import; grooming behavior; habituation; hippocampus development; intracellular protein transport; learning; locomotory behavior; maternal behavior; mating behavior; memory; muscle contraction; negative regulation of cell migration; negative regulation of circadian sleep/wake cycle, sleep; negative regulation of protein kinase activity; operant conditioning; orbitofrontal cortex development; peristalsis; phosphatidylinositol catabolic process; phosphatidylinositol metabolic process; positive regulation of adenylate cyclase activity; positive regulation of cAMP biosynthetic process; positive regulation of cell migration; positive regulation of membrane potential; positive regulation of release of sequestered calcium ion into cytosol; positive regulation of synaptic transmission, glutamatergic; prepulse inhibition; protein import into nucleus; regulation of dopamine metabolic process; regulation of ion transport; regulation of long-term neuronal synaptic plasticity; regulation of vasoconstriction; response to activity; response to amino acid stimulus; response to amphetamine; response to cocaine; response to drug; response to estradiol stimulus; response to ethanol; response to food; response to morphine; response to nicotine; response to organic cyclic substance; response to organic nitrogen; response to retinoic acid; response to steroid hormone stimulus; sensitization; social behavior; startle response; striatum development; synaptic transmission, dopaminergic; synaptogenesis; thermoregulation; transmission of nerve impulse; vasodilation; visual learning
NCBI Summary:	induces dopamine sensitive adenylate cyclase activation; plays a role in regulating food intake [RGD, Feb 2006]
UniProt Code:	P18901
NCBI GenInfo Identifier:	118229
NCBI Gene ID:	24316
NCBI Accession:	P18901.2
UniProt Secondary Accession:	P18901,P21669,
UniProt Related Accession:	P18901
Molecular Weight:	49,428 Da
NCBI Full Name:	D(1A) dopamine receptor
NCBI Synonym Full Names:	dopamine receptor D1
NCBI Official Symbol:	Drd1
NCBI Official Synonym Symbols:	D1a; Drd-1; Drd1a
NCBI Protein Information:	D(1A) dopamine receptor
UniProt Protein Name:	D(1A) dopamine receptor
UniProt Synonym Protein Names:	Dopamine D1 receptor
Protein Family:	D(1A) dopamine receptor
UniProt Gene Name:	Drd1
UniProt Entry Name:	DRD1_RAT

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 clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum 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 anti-coagulant. 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 & homogenizein 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.