Rat DRD4 (Dopamine Receptor D4) CLIA Kit (RTES00186)

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SKU:
RTES00186
€649

Description

ELISA Kit Technical ManualMSDS

Rat DRD4 (Dopamine Receptor D4) CLIA Kit

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Rat DRD4 . Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Rat DRD4 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Rat DRD4, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Rat DRD4. The concentration of Rat DRD4 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityRat
Detection methodChemiluminescence
Detection range31.25-2000 pg/mL
Sensitivity18.75 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Rat DRD4 in samples. No significant cross-reactivity or interference between Rat DRD4 and analogues was observed.

Typical Data

As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration
(pg/mL)
RLU Average Corrected
200053960
57482
5572155693
100023777
24171
2397423946
50011990
10074
1103211004
2505051
5537
52945266
1252765
2451
26082580
62.51324
1298
13111283
31.25638
710
674646
027
29
28--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat DRD4 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat DRD4 were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)94.73214.58774.0898.17195.37839.44
Standard deviation7.9317.4077.4912.5619.7188.56
C V (%)8.378.1110.0112.7910.0910.55

Recovery

The recovery of Rat DRD4 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type Range (%) Average Recovery (%)
Serum (n=5)100-114107
EDTA plasma (n=5)94-108101
Cell culture media (n=5)87-10094

Linearity

Samples were spiked with high concentrations of Rat DRD4 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

    Serum (n=5) EDTA plasma (n=5) Cell culture media (n=5)
1:2Range (%)91-10396-10691-107
Average (%)9710198
1:4Range (%)87-99102-11595-110
Average (%)92109100
1:8Range (%)95-10789-104103-116
Average (%)10196109
1:16Range (%)86-9985-9693-105
Average (%)919199

Kit Components & Storage

An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item Specifications Storage
Micro CLIA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Rat DRD4 (Dopamine Receptor D4) CLIA Kit (RTES00186) Assay procedure

    1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids.) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37°C.
    7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
    11. Determine the RLU value of each well immediately.

Rat DRD4 (Dopamine Receptor D4) CLIA Kit (RTES00186) Protein Information

UniProt Protein Function:DRD4: Dopamine receptor responsible for neuronal signaling in the mesolimbic system of the brain, an area of the brain that regulates emotion and complex behavior. Its activity is mediated by G proteins which inhibit adenylyl cyclase. Belongs to the G-protein coupled receptor 1 family.
UniProt Protein Details:

Protein type:GPCR, family 1; Receptor, GPCR; Membrane protein, multi-pass; Membrane protein, integral

Cellular Component: axon; cell cortex; cell soma; cytoplasm; dendrite; dendritic spine; integral to plasma membrane; membrane; nerve terminal; plasma membrane; terminal button; vesicle membrane

Molecular Function:dopamine binding; dopamine D1 receptor-like receptor activity; dopamine D2 receptor-like receptor activity; dopamine receptor activity; drug binding; identical protein binding; protein binding; SH3 domain binding

Biological Process: activation of MAPK activity; adult locomotory behavior; arachidonic acid secretion; associative learning; behavioral fear response; behavioral response to cocaine; circadian rhythm; dopamine receptor signaling pathway; dopamine receptor, adenylate cyclase inhibiting pathway; fear response; locomotory behavior; negative regulation of adenylate cyclase activity; negative regulation of cAMP biosynthetic process; negative regulation of protein secretion; olfactory learning; photoperiodism; positive regulation of kinase activity; positive regulation of sodium:hydrogen antiporter activity; regulation of calcium-mediated signaling; regulation of circadian rhythm; regulation of dopamine metabolic process; regulation of inhibitory postsynaptic membrane potential; regulation of neurotransmitter secretion; regulation of synaptic transmission; response to amphetamine; response to steroid hormone stimulus; retina development in camera-type eye; short-term memory; synaptic transmission, dopaminergic

NCBI Summary:may play a role in exploratory behavior and motor coordination; may be involved in lesion induced hyperactivity [RGD, Feb 2006]
UniProt Code:P30729
NCBI GenInfo Identifier:6226851
NCBI Gene ID:25432
NCBI Accession:P30729.2
UniProt Secondary Accession:P30729,Q62610,
UniProt Related Accession:P30729
Molecular Weight:41,294 Da
NCBI Full Name:D(4) dopamine receptor
NCBI Synonym Full Names:dopamine receptor D4
NCBI Official Symbol:Drd4  
NCBI Official Synonym Symbols:D4RA  
NCBI Protein Information:D(4) dopamine receptor
UniProt Protein Name:D(4) dopamine receptor
UniProt Synonym Protein Names:D(2C) dopamine receptor; Dopamine D4 receptor
Protein Family:D(4) dopamine receptor
UniProt Gene Name:Drd4  
UniProt Entry Name:DRD4_RAT
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Additional Information

Reactivity:
Rat
ELISA Type:
CLIA
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