Description
Human Bile acid receptor (NR1H4) ELISA Kit
The Human Bile Acid Receptor NR1H4 ELISA Kit from AssayGenie is specifically designed for the accurate measurement of bile acid receptor NR1H4 levels in human samples including serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring precise and reliable results for a variety of research applications.The bile acid receptor NR1H4, also known as Farnesoid X Receptor (FXR), plays a crucial role in regulating bile acid metabolism and homeostasis in the liver and intestine. Dysregulation of NR1H4 has been linked to various metabolic disorders, including liver diseases, obesity, and diabetes.
By accurately quantifying NR1H4 levels, researchers can gain valuable insights into the mechanisms underlying these conditions and explore potential therapeutic targets.With its easy-to-use protocol and robust performance, the Human Bile Acid Receptor NR1H4 ELISA Kit is an essential tool for studying NR1H4 function and its impact on metabolic health. Trust AssayGenie for accurate and dependable results in your research endeavors.
| Product Name: | Human Bile acid receptor (NR1H4) ELISA Kit |
| SKU: | HUEB2126 |
| Size: | 96T |
| Target: | Human Bile acid receptor (NR1H4) |
| Synonyms: | 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, BAR, FXR, HRR1, RIP14 |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 0.156-10ng/mL |
| Sensitivity: | 0.078ng/mL |
| Intra CV: | 6.1% | ||||||||||||||||||||
| Inter CV: | 8.9% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Isoform 4: Promotes transcriptional activation of target genes ABCB11/BSEP (inducible by unconjugated CDCA, ACA and DCA), NR0B2/SHP (inducible by unconjugated CDCA, ACA and DCA), SLC51B/OSTB (inducible by unconjugated CDCA and DCA) and FABP6/IBAP; most efficient isoform compared to isoforms 1 to 3; not inducible by taurine- and glycine-amidated CDCA. |
| Uniprot: | Q96RI1 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human Bile acid receptor |
| Sub Unit: | Binds DNA predominantly as a heterodimer with RXRA. After activation by agonist binding interacts with coactivators. Interacts with NCOA1, NCOA2, PPARGC1A, CARM1, SETD7, PRMT1, GPS2, SMARCA4 and MED1 (PubMed:15202934, PubMed:14684751, PubMed:15187081, PubMed:15471871, PubMed:15911693, PubMed:17895379, PubMed:18755856, PubMed:19805516, PubMed:23462506, PubMed:12718892, PubMed:12718893, PubMed:18621523, PubMed:18391212, PubMed:19410460, PubMed:19586769). Interacts with EP300 and SMARCD1 (By similarity). Interacts with XRCC5 and XRCC6; decreasing NR1H4/FXR transactivation activity towards ABCB11/BSEP (PubMed:19833092). Interacts with PAGR1 AND NCOA6; indicative for an association with an MLL2/MLL3 complex (ASCOM) (PubMed:19556342). |
| Research Area: | Cardiovascular |
| Subcellular Location: | Isoform 4 Nucleus |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| 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 |
| 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. |
