Bovine Signalling ELISA Kits

Bovine Bile acid receptor (NR1H4) ELISA Kit

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SKU:
BOEB0959
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q3SZL0
ELISA Type:
Sandwich
Reactivity:
Bovine

Description

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Product Name:Bovine Bile acid receptor (NR1H4) ELISA Kit
Product Code:BOEB0959
Alias:Bile acid receptor, Farnesoid X-activated receptor, Farnesol receptor HRR-1, Nuclear receptor subfamily 1 group H member 4, NR1H4
Uniprot:Q3SZL0
Reactivity:Bovine
Range:Please contact us for more information
Detection Method:Sandwich
Size:96 Assay
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:Ligand-activated transcription factor. Receptor for bile acids (BAs) such as chenodeoxycholic acid (CDCA), lithocholic acid, deoxycholic acid (DCA) and allocholic acid (ACA). Plays a essential role in BA homeostasis through the regulation of genes involved in BA synthesis, conjugation and enterohepatic circulation. Also regulates lipid and glucose homeostasis and is involved innate immune response. The FXR-RXR heterodimer binds predominantly to farnesoid X receptor response elements (FXREs) containing two inverted repeats of the consensus sequence 5'-AGGTCA-3' in which the monomers are spaced by 1 nucleotide (IR-1) but also to tandem repeat DR1 sites with lower affinity, and can be activated by either FXR or RXR-specific ligands. It is proposed that monomeric nuclear receptors such as NR5A2/LRH-1 bound to coregulatory nuclear responsive element (NRE) halfsites located in close proximity to FXREs modulate transcriptional activity. In the liver activates transcription of the corepressor NR0B2 thereby indirectly inhibiting CYP7A1 and CYP8B1 (involved in BA synthesis) implicating at least in part histone demethylase KDM1A resulting in epigenomic repression, and SLC10A1/NTCP (involved in hepatic uptake of conjugated BAs). Activates transcription of the repressor MAFG (involved in regulation of BA synthesis). Activates transcription of SLC27A5/BACS and BAAT (involved in BA conjugation), ABCB11/BSEP (involved in bile salt export) by directly recruiting histone methyltransferase CARM1, and ABCC2/MRP2 (involved in secretion of conjugated BAs) and ABCB4 (involved in secretion of phosphatidylcholine in the small intestine). Activates transcription of SLC27A5/BACS and BAAT (involved in BA conjugation), ABCB11/BSEP (involved in bile salt export) by directly recruiting histone methyltransferase CARM1, and ABCC2/MRP2 (involved in secretion of conjugated BAs) and ABCB4 (involved in secretion of phosphatidylcholine in the small intestine). In the intestine activates FGF19 expression and secretion leading to hepatic CYP7A1 repression. The function also involves the coordinated induction of hepatic KLB/beta-klotho expression. Regulates transcription of liver UGT2B4 and SULT2A1 involved in BA detoxification; binding to the UGT2B4 promoter seems to imply a monomeric transactivation independent of RXRA. Modulates lipid homeostasis by activating liver NR0B2/SHP-mediated repression of SREBF1 (involved in de novo lipogenesis), expression of PLTP (involved in HDL formation), SCARB1 (involved in HDL hepatic uptake), APOE, APOC1, APOC4, PPARA (involved in beta-oxidation of fatty acids), VLDLR and SDC1 (involved in the hepatic uptake of LDL and IDL remnants), and inhibiting expression of MTTP (involved in VLDL assembly). Increases expression of APOC2 (promoting lipoprotein lipase activity implicated in triglyceride clearance). Transrepresses APOA1 involving a monomeric competition with NR2A1 for binding to a DR1 element. Also reduces triglyceride clearance by inhibiting expression of ANGPTL3 and APOC3 (both involved in inhibition of lipoprotein lipase). Involved in glucose homeostasis by modulating hepatic gluconeogenesis through activation of NR0B2/SHP-mediated repression of respective genes. Modulates glycogen synthesis (inducing phosphorylation of glycogen synthase kinase-3). Modulates glucose-stimulated insulin secretion and is involved in insulin resistance. Involved in intestinal innate immunity. Plays a role in protecting the distal small intestine against bacterial overgrowth and preservation of the epithelial barrier. Down-regulates inflammatory cytokine expression in several types of immune cells including macrophages and mononuclear cells. Mediates trans-repression of TLR4-induced cytokine expression; the function seems to require its sumoylation and prevents N-CoR nuclear receptor corepressor clearance from target genes such as IL1B and NOS2. Involved in the TLR9-mediated protective mechanism in intestinal inflammation. Plays an anti-inflammatory role in liver inflammation; proposed to inhibit proinflammatory (but not antiapoptotic) NF-kappa-B signaling.
UniProt Protein Details:
NCBI Summary:
UniProt Code:Q3SZL0
NCBI GenInfo Identifier:77736361
NCBI Gene ID:540528
NCBI Accession:NP_001029880.1
UniProt Secondary Accession:Q3SZL0
UniProt Related Accession:Q3SZL0
Molecular Weight:55,460 Da
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:
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
Protein Family:Bile acid receptor
UniProt Gene Name:NR1H4  
UniProt Entry Name:
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.
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