Human RARA ELISA Kit (HUEB2335)- High Sensitivity

Product Name:	Human Retinoic acid receptor alpha (RARA) ELISA Kit
SKU:	HUEB2335
Size:	96T
Target:	Human Retinoic acid receptor alpha (RARA)
Synonyms:	Nuclear receptor subfamily 1 group B member 1, RAR-alpha, NR1B1
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.312-20ng/mL
Sensitivity:	0.13ng/mL

Intra CV:

4.4%

Inter CV:

9.7%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	109-120%	99-111%	103-113%	82-92%
EDTA Plasma(N=5)	96-105%	94-104%	91-102%	103-112%
Heparin Plasma(N=5)	117-127%	104-116%	82-92%	102-110%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	101	95-107
Plasma	103	97-109

Function:

Receptor for retinoic acid. Retinoic acid receptors bind as heterodimers to their target response elements in response to their ligands, all-trans or 9-cis retinoic acid, and regulate gene expression in various biological processes. The RXR/RAR heterodimers bind to the retinoic acid response elements (RARE) composed of tandem 5'-AGGTCA-3' sites known as DR1-DR5. In the absence of ligand, the RXR-RAR heterodimers associate with a multiprotein complex containing transcription corepressors that induce histone acetylation, chromatin condensation and transcriptional suppression. On ligand binding, the corepressors dissociate from the receptors and associate with the coactivators leading to transcriptional activation. RARA plays an essential role in the regulation of retinoic acid-induced germ cell development during spermatogenesis. Has a role in the survival of early spermatocytes at the beginning prophase of meiosis. In Sertoli cells, may promote the survival and development of early meiotic prophase spermatocytes. In concert with RARG, required for skeletal growth, matrix homeostasis and growth plate function (By similarity). Regulates expression of target genes in a ligand-dependent manner by recruiting chromatin complexes containing KMT2E/MLL5. Mediates retinoic acid-induced granulopoiesis.

Uniprot:	P10276
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Retinoic acid receptor alpha
Sub Unit:	Heterodimer; with RXRA. Binds DNA preferentially as a heterodimer. Interacts with CDK7 (By similarity). Interacts with coactivators NCOA3 and NCOA6. Interacts with NCOA7; the interaction requires ligand-binding. Interacts with KMT2E/MLL5. Interacts (via the ligand-binding domain) with PRAME; the interaction is ligand (retinoic acid)-dependent. Interacts with AKT1; the interaction phosphorylates RARA and represses transactivation. Interacts with PRKAR1A; the interaction negatively regulates RARA transcriptional activity. Interacts with NCOR1 and NCOR2. Interacts with PRMT2. Interacts with LRIF1. Interacts with ASXL1 and NCOA1. Interacts with ACTN4.
Research Area:	Cancer
Subcellular Location:	Nucleus Cytoplasm Nuclear localization depends on ligand binding, phosphorylation and sumoylation. Transloaction to the nucleus in the absence of ligand is dependent on activation of PKC and the downstream MAPK phosphorylation.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

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

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

ELISA	Antibodies
Human Retinoic Acid Receptor Alpha / RAR alpha ELISA Kit	Anti-RARA Antibody (CAB0370)
Human RAR alpha (Retinoic Acid Receptor Alpha) CLIA Kit (HUES01255)	Anti-RARA Antibody (CAB14057)
Retinoic Acid Receptor alpha (Phospho-Ser77) Transcription Factor Activity Assay	Anti-Retinoic Acid Receptor alpha Antibody (CAB19551)
Retinoic Acid Receptor alpha Transcription Factor Activity Assay	Anti-RARA Antibody (CAB7420)
	Phospho-RARA (S77) Antibody (PACO04881)