Mouse DAT (Dopamine Transporter) ELISA Kit
This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Mouse DAT . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Mouse 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 Mouse DAT, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Mouse DAT. The concentration of Mouse DAT in samples can be calculated by comparing the OD of the samples to the standard curve.
|Detection Range||7.81-500 pg/mL|
|Sample Volume Required Per Well||100uL|
|Sample Type||Serum, plasma and other biological fluids|
This kit recognizes Mouse DAT in samples. No significant cross-reactivity or interference between Mouse DAT and analogues was observed.
As the OD 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.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse DAT were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse DAT were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||5.32||4.43||4.35||6.63||4.74||5.29|
The recovery of Mouse DAT spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
|Sample Type||Range (%)||Average Recovery (%)|
|EDTA plasma (n=5)||84-97||91|
|Cell culture media (n=5)||85-99||91|
Samples were spiked with high concentrations of Mouse 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)|
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.
|Micro ELISA Plate(Dismountable)||8 wells X12 strips||-20'C, 6 months|
|Reference Standard||2 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 Diluent||1 vial, 20 mL||4'C, 6 months|
|Biotinylated Detection Ab Diluent||1 vial, 14 mL|
|HRP Conjugate Diluent||1 vial, 14 mL|
|Concentrated Wash Buffer (25X)||1 vial, 30 mL|
|Substrate Reagent||1 vial, 10 mL||4'C(shading light)|
|Stop Solution||1 vial, 10 mL||4'C|
|Plate Sealer||5 pieces|
|Product Description||1 copy|
|Certificate of Analysis||1 copy|
Mouse DAT (Dopamine Transporter) ELISA Kit (MOES00969) 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 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
- 11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- 12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
Mouse DAT (Dopamine Transporter) ELISA Kit (MOES00969) 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:Transporter, SLC family; Transporter; Membrane protein, multi-pass; Membrane protein, integral
Chromosomal Location of Human Ortholog: 5p15.3
Cellular Component: cell soma; axon; integral to plasma membrane; cytoplasm; integral to membrane; plasma membrane
Molecular Function:protein binding; protease binding; dopamine binding; monoamine transmembrane transporter activity; protein complex binding; protein N-terminus binding; dopamine:sodium symporter activity; drug binding; dopamine transmembrane transporter activity; receptor binding
Biological Process: lactation; response to drug; response to nicotine; dopamine transport; response to cAMP; prepulse inhibition; dopamine catabolic process; regulation of dopamine metabolic process; monoamine transport; sensory perception of smell; locomotory behavior; dopamine biosynthetic process; positive regulation of multicellular organism growth; response to cocaine; synaptic transmission; response to ethanol; neurotransmitter transport; adenohypophysis development; response to iron ion; transmembrane transport; aging; neurotransmitter biosynthetic process
Disease: Parkinsonism-dystonia, Infantile; Tobacco Addiction, Susceptibility To
|NCBI Summary:||This gene encodes a dopamine transporter which is a member of the sodium- and chloride-dependent neurotransmitter transporter family. The 3' UTR of this gene contains a 40 bp tandem repeat, referred to as a variable number tandem repeat or VNTR, which can be present in 3 to 11 copies. Variation in the number of repeats is associated with idiopathic epilepsy, attention-deficit hyperactivity disorder, dependence on alcohol and cocaine, susceptibility to Parkinson disease and protection against nicotine dependence.[provided by RefSeq, Nov 2009]|
|NCBI GenInfo Identifier:||4507041|
|NCBI Gene ID:||6531|
|UniProt Secondary Accession:||Q61327,Q61327, P23977,|
|UniProt Related Accession:||Q01959|
|Molecular Weight:||68,495 Da|
|NCBI Full Name:||sodium-dependent dopamine transporter|
|NCBI Synonym Full Names:||solute carrier family 6 (neurotransmitter transporter, dopamine), member 3|
|NCBI Official Symbol:||SLC6A3|
|NCBI Official Synonym Symbols:||DAT; DAT1; PKDYS|
|NCBI Protein Information:||sodium-dependent dopamine transporter; DA transporter; solute carrier family 6 member 3|
|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|
|UniProt Entry Name:||SC6A3_HUMAN|