Product Name:	Human RXRa (Retinoid X Receptor Alpha) ELISA Kit
Product Code:	HUFI03412
Size:	96 Assays
Alias:	RXRa ELISA Kit, Retinoid X Receptor Alpha ELISA Kit, Retinoic acid receptor RXR-alpha ELISA Kit, Nuclear receptor subfamily 2 group B member 1 ELISA Kit, NR2B1 ELISA Kit
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human RXRa (Retinoid X Receptor Alpha) concentrations in serum plasma and other biological fluids.
Sensitivity:	< 0.188ng/ml
Range:	0.313-20ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human RXRa (Retinoid X Receptor Alpha) and the recovery rates were calculated by comparing the measured value to the expected amount of Human RXRa (Retinoid X Receptor Alpha) in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-105	98
EDTA plasma(n=5)	85-94	92
heparin plasma(n=5)	93-105	99

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human RXRa (Retinoid X Receptor Alpha) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	96-102%	85-101%	85-98%
EDTA plasma(n=5)	83-100%	89-94%	84-91%
UFH plasma(n=5)	84-94%	83-100%	80-97%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	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

UniProt Protein Function:	RXRA: 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 RAR/RXR heterodimers bind to the retinoic acid response elements (RARE) composed of tandem 5'-AGGTCA-3' sites known as DR1-DR5. The high affinity ligand for RXRs is 9-cis retinoic acid. RXRA serves as a common heterodimeric partner for a number of nuclear receptors. 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. The RXRA/PPARA heterodimer is required for PPARA transcriptional activity on fatty acid oxidation genes such as ACOX1 and the P450 system genes. Homodimer. Heterodimer with RARA; required for ligand- dependent retinoic acid receptor transcriptional activity. Heterodimer with PPARA (via the leucine-like zipper in the LBD); the interaction is required for PPARA transcriptional activity. Also heterodimerizes with PPARG. Interacts with NCOA3 and NCOA6 coactivators. Interacts with FAM120B. Interacts with PELP1, SENP6, SFPQ, DNTTIP2 and RNF8. Interacts (via the DNA binding domain) with HCV core protein; the interaction enhances the transcriptional activities of the RXRA/RARA and the RXRA/PPARA heterodimers. Interacts with PRMT2. Interacts with ASXL1 and NCOA1. Highly expressed in liver, also found in lung, kidney and heart. Belongs to the nuclear hormone receptor family. NR2 subfamily.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; DNA-binding; Nuclear receptor Chromosomal Location of Human Ortholog: 9q34.3 Cellular Component: nucleoplasm; nuclear chromatin; nucleus; receptor complex Molecular Function:ligand-dependent nuclear receptor activity; zinc ion binding; chromatin DNA binding; transcription coactivator activity; protein binding; enzyme binding; DNA binding; vitamin D receptor binding; retinoid-X receptor activity; protein heterodimerization activity; sequence-specific DNA binding; steroid hormone receptor activity; retinoic acid receptor activity; transcription factor activity Biological Process: retinoic acid receptor signaling pathway; transcription initiation from RNA polymerase II promoter; cholesterol metabolic process; camera-type eye development; vitamin metabolic process; response to retinoic acid; in utero embryonic development; ventricular cardiac muscle cell differentiation; cellular lipid metabolic process; negative regulation of transcription from RNA polymerase II promoter; positive regulation of translational initiation by iron; protein homotetramerization; virus-host interaction; ventricular cardiac muscle morphogenesis; positive regulation of transcription from RNA polymerase II promoter; gene expression; steroid hormone mediated signaling; transmembrane transport; embryo implantation; maternal placenta development
NCBI Summary:	Retinoid X receptors (RXRs) and retinoic acid receptors (RARs) are nuclear receptors that mediate the biological effects of retinoids by their involvement in retinoic acid-mediated gene activation. These receptors function as transcription factors by binding as homodimers or heterodimers to specific sequences in the promoters of target genes. The protein encoded by this gene is a member of the steroid and thyroid hormone receptor superfamily of transcriptional regulators. Alternative splicing of this gene results in multiple transcript variants. [provided by RefSeq, May 2014]
UniProt Code:	P19793
NCBI GenInfo Identifier:	133701
NCBI Gene ID:	6256
NCBI Accession:	P19793.1
UniProt Secondary Accession:	P19793,Q2NL52, Q2V504, B3KY83,
UniProt Related Accession:	P19793
Molecular Weight:	41,060 Da
NCBI Full Name:	Retinoic acid receptor RXR-alpha
NCBI Synonym Full Names:	retinoid X receptor, alpha
NCBI Official Symbol:	RXRA
NCBI Official Synonym Symbols:	NR2B1
NCBI Protein Information:	retinoic acid receptor RXR-alpha
UniProt Protein Name:	Retinoic acid receptor RXR-alpha
UniProt Synonym Protein Names:	Nuclear receptor subfamily 2 group B member 1; Retinoid X receptor alpha
Protein Family:	Retinoic acid receptor
UniProt Gene Name:	RXRA
UniProt Entry Name:	RXRA_HUMAN

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

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37 °C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37 °C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37 °C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37 °C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

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.