Rat DAT (Dopamine Transporter) CLIA Kit (RTES00187)

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RTES00187
€649

Description

ELISA Kit Technical ManualMSDS

Rat DAT (Dopamine Transporter) 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 DAT . 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 DAT 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 DAT, 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 DAT. The concentration of Rat DAT 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 range62.50-4000 pg/mL
Sensitivity37.50 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Rat DAT in samples. No significant cross-reactivity or interference between Rat DAT 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
400054393
63769
5908159046
200027326
28392
2785927824
100014167
12801
1348413449
5006519
6691
66056570
2503397
3089
32433208
1251693
1469
15811546
62.50717
793
755720
034
36
35--

Precision

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

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

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)190.07468.921922.87205.93497.771874.53
Standard deviation19.3334.79158.0618.0852.51133.47
C V (%)10.177.428.228.7810.557.12

Recovery

The recovery of Rat DAT 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)99-114105
EDTA plasma (n=5)89-10495
Cell culture media (n=5)100-115106

Linearity

Samples were spiked with high concentrations of Rat DAT 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 (%)92-10787-10097-112
Average (%)9893105
1:4Range (%)89-10394-10790-102
Average (%)9510196
1:8Range (%)103-11995-10991-105
Average (%)11010299
1:16Range (%)98-11399-11791-104
Average (%)10410798

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 DAT (Dopamine Transporter) CLIA Kit (RTES00187) 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 DAT (Dopamine Transporter) CLIA Kit (RTES00187) Protein Information

UniProt Protein Function:DAT: Amine transporter. Terminates the action of dopamine by its high affinity sodium-dependent reuptake into presynaptic terminals. Defects in SLC6A3 are the cause of dystonia-parkinsonism infantile (DYTPRI). It is a neurodegenerative disorder characterized by infantile onset of parkinsonism and dystonia. Other neurologic features include global developmental delay, bradikinesia and pyramidal tract signs. Belongs to the sodium:neurotransmitter symporter (SNF) (TC 2.A.22) family. SLC6A3 subfamily.
UniProt Protein Details:

Protein type:Membrane protein, integral; Membrane protein, multi-pass; Transporter; Transporter, SLC family

Chromosomal Location of Human Ortholog: 1p11

Cellular Component: axon; cell soma; cell surface; flotillin complex; integral component of plasma membrane; membrane raft; neuron projection; plasma membrane; presynapse

Molecular Function:dopamine binding; dopamine transmembrane transporter activity; dopamine:sodium symporter activity; drug binding; metal ion binding; monoamine transmembrane transporter activity; protease binding; protein complex binding; protein N-terminus binding; protein phosphatase 2A binding; receptor binding

Biological Process: adenohypophysis development; aging; ammonium transmembrane transport; dopamine biosynthetic process; dopamine catabolic process; dopamine transport; dopamine uptake; lactation; locomotory behavior; monoamine transport; positive regulation of multicellular organism growth; prepulse inhibition; regulation of dopamine metabolic process; response to cAMP; response to cocaine; response to drug; response to ethanol; response to iron ion; response to nicotine; response to organic cyclic compound; sensory perception of smell

NCBI Summary:acts as a sodium-dependent dopamine transporter; may play a role in regulation of dopamine metabolism and signaling [RGD, Feb 2006]
UniProt Code:P23977
NCBI GenInfo Identifier:7106413
NCBI Gene ID:24898
NCBI Accession:NP_036826.1
UniProt Related Accession:P23977
Molecular Weight:68,746 Da
NCBI Full Name:sodium-dependent dopamine transporter
NCBI Synonym Full Names:solute carrier family 6 member 3
NCBI Official Symbol:Slc6a3  
NCBI Official Synonym Symbols:Dat1  
NCBI Protein Information:sodium-dependent dopamine transporter
UniProt Protein Name:Sodium-dependent dopamine transporter
UniProt Synonym Protein Names:Solute carrier family 6 member 3
Protein Family:D-alanine aminotransferase
UniProt Gene Name:Slc6a3  
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Additional Information

Reactivity:
Rat
ELISA Type:
CLIA
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