Human THR alpha (Thyroid Hormone Receptor Alpha) ELISA Kit (HUES03285)

SKU:
HUES03285
€539

Description

ELISA Kit Technical ManualMSDS

Human THR alpha (Thyroid Hormone Receptor Alpha) 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 THR alpha . 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 THR alpha 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 THR alpha, 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 THR alpha. The concentration of Human THR alpha 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 Range62.50-4000 pg/mL
Sensitivity37.50 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human THR alpha in samples. No significant cross-reactivity or interference between Human THR alpha 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
(pg/mL)
O.D Average Corrected
40002.407
2.417
2.4122.342
20001.522
1.552
1.5371.467
10000.872
0.838
0.8550.785
5000.465
0.475
0.470.4
2500.261
0.239
0.250.18
1250.171
0.159
0.1650.095
62.500.109
0.129
0.1190.049
00.063
0.077
0.07--

Precision

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

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

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)190.76383.851784.26176.77380.871730.36
Standard deviation9.9218.5092.079.5617.1467.66
C V (%)5.204.825.165.414.503.91

Recovery

The recovery of Human THR alpha 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)95-106100
EDTA plasma (n=5)94-106101
Cell culture media (n=5)88-9993

Linearity

Samples were spiked with high concentrations of Human THR alpha 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 (%)91-10697-11197-114
Average (%)97103105
1:4Range (%)91-10682-9284-95
Average (%)978789
1:8Range (%)89-10184-9685-98
Average (%)948992
1:16Range (%)90-10479-9285-97
Average (%)968691

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 THR alpha (Thyroid Hormone Receptor Alpha) ELISA Kit (HUES03285) 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 THR alpha (Thyroid Hormone Receptor Alpha) ELISA Kit (HUES03285) Protein Information

UniProt Protein Function:TR-alpha: nuclear hormone receptor and transcription factor. High affinity receptor for triiodothyronine. Interacts with SRC-3 and NCOA6 coactivators, leading to a strong increase of transcription of target genes. Four splice variant isoforms have been described.
UniProt Protein Details:

Protein type:DNA-binding; Nuclear receptor; Transcription factor

Chromosomal Location of Human Ortholog: 17q11.2

Cellular Component: nucleoplasm; cytosol; nucleus

Molecular Function:protein domain specific binding; protein binding; zinc ion binding; sequence-specific DNA binding; TATA-binding protein binding; chromatin DNA binding; protein complex binding; steroid hormone receptor activity; thyroid hormone receptor activity; transcription factor activity; transcription factor binding

Biological Process: transcription from RNA polymerase II promoter; transcription initiation from RNA polymerase II promoter; negative regulation of transcriptional preinitiation complex assembly; ossification; cytoplasmic sequestering of transcription factor; intracellular receptor-mediated signaling pathway; hormone-mediated signaling; positive regulation of female receptivity; regulation of myeloid cell apoptosis; regulation of lipid catabolic process; regulation of heart contraction; regulation of transcription from RNA polymerase II promoter; female courtship behavior; learning and/or memory; thyroid gland development; erythrocyte differentiation; steroid hormone mediated signaling; gene expression; positive regulation of transcription from RNA polymerase II promoter; response to cold; negative regulation of transcription, DNA-dependent; cartilage condensation

Disease: Hypothyroidism, Congenital, Nongoitrous, 6

NCBI Summary:The protein encoded by this gene is a nuclear hormone receptor for triiodothyronine. It is one of the several receptors for thyroid hormone, and has been shown to mediate the biological activities of thyroid hormone. Knockout studies in mice suggest that the different receptors, while having certain extent of redundancy, may mediate different functions of thyroid hormone. Alternatively spliced transcript variants encoding distinct isoforms have been reported. [provided by RefSeq, Jul 2008]
UniProt Code:P10827
NCBI GenInfo Identifier:135705
NCBI Gene ID:7067
NCBI Accession:P10827.1
UniProt Secondary Accession:P10827,P21205, Q8N6A1, Q96H73, A8K3B5,
UniProt Related Accession:P10827
Molecular Weight:50,465 Da
NCBI Full Name:Thyroid hormone receptor alpha
NCBI Synonym Full Names:thyroid hormone receptor, alpha
NCBI Official Symbol:THRA
NCBI Official Synonym Symbols:AR7; EAR7; ERBA; CHNG6; ERBA1; NR1A1; THRA1; THRA2; ERB-T-1; c-ERBA-1
NCBI Protein Information:thyroid hormone receptor alpha; EAR-7; c-erbA-alpha; ERBA-related 7; V-erbA-related protein 7; triiodothyronine receptor; nuclear receptor subfamily 1 group A member 1; thyroid normone nuclear receptor alpha variant 1; thyroid hormone receptor, alpha (erythroblastic leukemia viral (v-erb-a) oncogene homolog, avian)
UniProt Protein Name:Thyroid hormone receptor alpha
UniProt Synonym Protein Names:Nuclear receptor subfamily 1 group A member 1; V-erbA-related protein 7; EAR-7; c-erbA-1; c-erbA-alpha
Protein Family:Thyroid hormone receptor
UniProt Gene Name:THRA
UniProt Entry Name:THA_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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