Human RAR alpha (Retinoic Acid Receptor Alpha) ELISA Kit (HUES03280)

SKU:
HUES03280
€539

Description

ELISA Kit Technical ManualMSDS

Human RAR alpha (Retinoic Acid 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 RAR 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 RAR 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 RAR 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 RAR alpha. The concentration of Human RAR 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 Range0.16-10 ng/mL
Sensitivity0.10 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human RAR alpha in samples. No significant cross-reactivity or interference between Human RAR 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
(ng/mL)
O.D Average Corrected
102.288
2.318
2.3032.235
51.518
1.576
1.5471.479
2.50.982
0.954
0.9680.9
1.250.417
0.435
0.4260.358
0.630.274
0.244
0.2590.191
0.320.176
0.164
0.170.102
0.160.118
0.122
0.120.052
00.064
0.072
0.068--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human RAR 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 RAR alpha were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.531.204.630.571.214.81
Standard deviation0.030.060.180.040.060.20
C V (%)5.665.003.897.024.964.16

Recovery

The recovery of Human RAR 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)89-10294
EDTA plasma (n=5)87-9994
Cell culture media (n=5)96-107101

Linearity

Samples were spiked with high concentrations of Human RAR 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 (%)94-10785-9888-104
Average (%)999295
1:4Range (%)90-10585-9881-93
Average (%)969188
1:8Range (%)92-10482-9686-98
Average (%)988892
1:16Range (%)87-9885-10088-101
Average (%)929195

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 RAR alpha (Retinoic Acid Receptor Alpha) ELISA Kit (HUES03280) 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 RAR alpha (Retinoic Acid Receptor Alpha) ELISA Kit (HUES03280) Protein Information

UniProt Protein Function:RARA: is a receptor for retinoic acid, a potent mammalian morphogen and teratogen that has profound effects on vertebrate development. RARA is a member of the nuclear receptor superfamily. Controls cell function by directly regulating gene expression. Its phosphorylation is crucial for transcriptional activity. Aberrations involving RARA may be a cause of acute promyelocytic leukemia. Two splice-variant isoforms have been described.
UniProt Protein Details:

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

Chromosomal Location of Human Ortholog: 17q21

Cellular Component: nucleoplasm; cell surface; cell soma; perinuclear region of cytoplasm; cytoplasm; dendrite; nuclear chromatin; nucleus; actin cytoskeleton

Molecular Function:protein domain specific binding; protein kinase B binding; retinoic acid binding; zinc ion binding; chromatin DNA binding; translation repressor activity, nucleic acid binding; transcription coactivator activity; phosphoinositide 3-kinase regulator activity; drug binding; alpha-actinin binding; transcription factor binding; protein binding; enzyme binding; protein heterodimerization activity; protein kinase A binding; steroid hormone receptor activity; mRNA 5'-UTR binding; retinoic acid receptor activity; transcription factor activity; transcription corepressor activity; receptor binding

Biological Process: retinoic acid receptor signaling pathway; limb development; prostate gland development; negative regulation of translational initiation; regulation of myelination; estrogen receptor signaling pathway; glandular epithelial cell development; positive regulation of transcription, DNA-dependent; ventricular cardiac muscle cell differentiation; regulation of synaptic plasticity; female pregnancy; negative regulation of transcription from RNA polymerase II promoter; signal transduction; protein amino acid phosphorylation; regulation of phosphoinositide 3-kinase activity; response to estradiol stimulus; response to vitamin A; negative regulation of granulocyte differentiation; germ cell development; positive regulation of interleukin-4 production; negative regulation of cell proliferation; Sertoli cell fate commitment; positive regulation of T-helper 2 cell differentiation; ureteric bud development; negative regulation of interferon-gamma production; positive regulation of cell proliferation; positive regulation of interleukin-13 production; transmembrane transport; positive regulation of interleukin-5 production; transcription initiation from RNA polymerase II promoter; response to retinoic acid; multicellular organism growth; positive regulation of cell cycle; positive regulation of binding; negative regulation of tumor necrosis factor production; liver development; embryonic camera-type eye development; positive regulation of phosphoinositide 3-kinase cascade; positive regulation of protein kinase B signaling cascade; response to ethanol; response to cytokine stimulus; neural tube closure; gene expression; spermatogenesis; positive regulation of transcription from RNA polymerase II promoter; steroid hormone mediated signaling; positive regulation of neuron differentiation; negative regulation of transcription, DNA-dependent; apoptotic cell clearance; negative regulation of apoptosis

Disease: Acute Promyelocytic Leukemia

NCBI Summary:This gene represents a nuclear retinoic acid receptor. The encoded protein, retinoic acid receptor alpha, regulates transcription in a ligand-dependent manner. This gene has been implicated in regulation of development, differentiation, apoptosis, granulopoeisis, and transcription of clock genes. Translocations between this locus and several other loci have been associated with acute promyelocytic leukemia. Alternatively spliced transcript variants have been found for this locus.[provided by RefSeq, Sep 2010]
UniProt Code:P10276
NCBI GenInfo Identifier:133483
NCBI Gene ID:5914
NCBI Accession:P10276.2
UniProt Secondary Accession:P10276,P78456, Q13440, Q13441, Q96S41, Q9NQS0, B8Y636
UniProt Related Accession:P10276
Molecular Weight:39,700 Da
NCBI Full Name:Retinoic acid receptor alpha
NCBI Synonym Full Names:retinoic acid receptor, alpha
NCBI Official Symbol:RARA
NCBI Official Synonym Symbols:RAR; NR1B1
NCBI Protein Information:retinoic acid receptor alpha; RAR-alpha; retinoic acid receptor, alpha polypeptide; nuclear receptor subfamily 1 group B member 1; retinoic acid nuclear receptor alpha variant 1; retinoic acid nuclear receptor alpha variant 2; nucleophosmin-retinoic acid receptor alpha fusion protein NPM-RAR long form
UniProt Protein Name:Retinoic acid receptor alpha
UniProt Synonym Protein Names:Nuclear receptor subfamily 1 group B member 1
Protein Family:Retinoic acid receptor
UniProt Gene Name:RARA
UniProt Entry Name:RARA_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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