Product Name:	Human NR1H4 / FXR ELISA Kit
Product Code:	HUFI01735
Size:	96 Assays
Alias:	NR1H4, Bile acid receptor, Retinoid X receptor-interacting protein 14, RXR-interacting protein 14, Nuclear receptor subfamily 1 group H member 4, Farnesoid X-activated receptor, Farnesol receptor, HRR-1, BAR, FXR, HRR1, RIP14
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human NR1H4 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human NR1H4 and the recovery rates were calculated by comparing the measured value to the expected amount of Human NR1H4 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	91-102	99
EDTA plasma(n=5)	89-102	96
UFH plasma(n=5)	86-105	96

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human NR1H4 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)	85-99%	85-100%	87-105%
EDTA plasma(n=5)	88-95%	82-98%	88-101%
UFH plasma(n=5)	85-93%	81-99%	83-100%

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

Q96RI1

UniProt Protein Function:	NR1H4: Ligand-activated transcription factor. Receptor for bile acids such as chenodeoxycholic acid, lithocholic acid and deoxycholic acid. Represses the transcription of the cholesterol 7-alpha-hydroxylase gene (CYP7A1) through the induction of NR0B2 or FGF19 expression, via two distinct mechanisms. Activates the intestinal bile acid-binding protein (IBABP). Activates the transcription of bile salt export pump ABCB11 by directly recruiting histone methyltransferase CARM1 to this locus. Heterodimer of NR1H4 and RXR. After activation by agonist binding, interacts with a coactivator, NCOA1 or NCOA2. Interacts with CARM1 and SMARD1. Belongs to the nuclear hormone receptor family. NR1 subfamily. 4 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:DNA-binding; Nuclear receptor Chromosomal Location of Human Ortholog: 12q23.1 Cellular Component: nucleoplasm Molecular Function:bile acid binding; ligand-dependent nuclear receptor activity; ligand-dependent nuclear receptor binding; protein binding; RNA polymerase II transcription factor activity, enhancer binding; sequence-specific DNA binding; transcription coactivator activity; transcription corepressor activity; transcription factor activity Biological Process: bile acid and bile salt transport; intracellular receptor-mediated signaling pathway; negative regulation of apoptosis; negative regulation of I-kappaB kinase/NF-kappaB cascade; negative regulation of transcription from RNA polymerase II promoter; positive regulation of transcription from RNA polymerase II promoter; signal transduction; toll-like receptor 4 signaling pathway; transcription initiation from RNA polymerase II promoter
NCBI Summary:	This gene encodes a ligand-activated transcription factor that shares structural features in common with nuclear hormone receptor family members. This protein functions as a receptor for bile acids, and when bound to bile acids, binds to DNA and regulates the expression of genes involved in bile acid synthesis and transport. Alternatively spliced transcript variants encoding different isoforms have been described. [provided by RefSeq, Feb 2016]
UniProt Code:	Q96RI1
NCBI GenInfo Identifier:	46577705
NCBI Gene ID:	9971
NCBI Accession:	Q96RI1.2
UniProt Secondary Accession:	Q96RI1,Q8NFP5, Q8NFP6, Q92943, A1L4K5, B7Z412, B7ZM06 F8VYG8,
UniProt Related Accession:	Q96RI1
Molecular Weight:	56kDa
NCBI Full Name:	Bile acid receptor
NCBI Synonym Full Names:	nuclear receptor subfamily 1 group H member 4
NCBI Official Symbol:	NR1H4
NCBI Official Synonym Symbols:	BAR; FXR; HRR1; HRR-1; PFIC5; RIP14
NCBI Protein Information:	bile acid receptor
UniProt Protein Name:	Bile acid receptor
UniProt Synonym Protein Names:	Farnesoid X-activated receptor; Farnesol receptor HRR-1; Nuclear receptor subfamily 1 group H member 4; Retinoid X receptor-interacting protein 14; RXR-interacting protein 14
Protein Family:	Bile acid receptor
UniProt Gene Name:	NR1H4

*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.