Human EAAT3(Excitatory Amino Acid Transporter 3)ELISA Kit (HUES03563)

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HUES03563
€539

Description

ELISA Kit Technical ManualMSDS

Human EAAT3(Excitatory Amino Acid Transporter 3)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 Human EAAT3 . 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 Human EAAT3 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 Human EAAT3, 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 Human EAAT3. The concentration of Human EAAT3 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range0.78-50 ng/mL
Sensitivity0.47 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human EAAT3 in samples. No significant cross-reactivity or interference between Human EAAT3 and analogues was observed.

Typical Data

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.

Concentration
(ng/mL)
O.D Average Corrected
502.451
2.481
2.4662.386
251.699
1.727
1.7131.633
12.50.99
0.982
0.9860.906
6.250.522
0.548
0.5350.455
3.130.297
0.267
0.2820.202
1.570.193
0.179
0.1860.106
0.780.124
0.144
0.1340.054
00.072
0.088
0.08--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human EAAT3 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human EAAT3 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)2.726.8117.022.487.2318.54
Standard deviation0.140.400.710.140.370.77
C V (%)5.155.874.175.655.124.15

Recovery

The recovery of Human EAAT3 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)86-9992
EDTA plasma (n=5)94-111101
Cell culture media (n=5)89-10196

Linearity

Samples were spiked with high concentrations of Human EAAT3 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 (%)88-9992-10696-111
Average (%)9398104
1:4Range (%)91-10882-9485-98
Average (%)998791
1:8Range (%)90-10483-9583-99
Average (%)978890
1:16Range (%)89-10282-9487-100
Average (%)958893

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 ELISA 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 Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human EAAT3(Excitatory Amino Acid Transporter 3)ELISA Kit (HUES03563) 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.

Human EAAT3(Excitatory Amino Acid Transporter 3)ELISA Kit (HUES03563) Protein Information

UniProt Protein Function:SLC1A1: Transports L-glutamate and also L- and D-aspartate. Essential for terminating the postsynaptic action of glutamate by rapidly removing released glutamate from the synaptic cleft. Acts as a symport by cotransporting sodium. Negatively regulated by ARL6IP5. Belongs to the sodium:dicarboxylate (SDF) symporter (TC 2.A.23) family. SLC1A1 subfamily.
UniProt Protein Details:

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

Chromosomal Location of Human Ortholog: 9p24

Cellular Component: membrane; integral to plasma membrane; apical plasma membrane; plasma membrane

Molecular Function:glutamate binding; cysteine transmembrane transporter activity; sodium:dicarboxylate symporter activity; glutamate:sodium symporter activity; L-glutamate transmembrane transporter activity

Biological Process: synaptic transmission; L-glutamate import; cysteine transport; ion transport; transmembrane transport; protein homooligomerization

Disease: Schizophrenia 18

NCBI Summary:This gene encodes a member of the high-affinity glutamate transporters that play an essential role in transporting glutamate across plasma membranes. In brain, these transporters are crucial in terminating the postsynaptic action of the neurotransmitter glutamate, and in maintaining extracellular glutamate concentrations below neurotoxic levels. This transporter also transports aspartate, and mutations in this gene are thought to cause dicarboxylicamino aciduria, also known as glutamate-aspartate transport defect. [provided by RefSeq, Mar 2010]
UniProt Code:P43005
NCBI GenInfo Identifier:1352332
NCBI Gene ID:6505
NCBI Accession:P43005.2
UniProt Secondary Accession:P43005,O75587, Q5VZ24, Q8N199, Q9UEW2,
UniProt Related Accession:P43005
Molecular Weight:57,100 Da
NCBI Full Name:Excitatory amino acid transporter 3
NCBI Synonym Full Names:solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, system Xag), member 1
NCBI Official Symbol:SLC1A1  
NCBI Official Synonym Symbols:DCBXA; EAAC1; EAAT3; SCZD18  
NCBI Protein Information:excitatory amino acid transporter 3; excitatory amino acid carrier 1; solute carrier family 1 member 1; neuronal and epithelial glutamate transporter; sodium-dependent glutamate/aspartate transporter 3
UniProt Protein Name:Excitatory amino acid transporter 3
UniProt Synonym Protein Names:Excitatory amino-acid carrier 1; Neuronal and epithelial glutamate transporter; Sodium-dependent glutamate/aspartate transporter 3; Solute carrier family 1 member 1
Protein Family:Excitatory amino acid transporter
UniProt Gene Name:SLC1A1  
UniProt Entry Name:EAA3_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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