Rat Farnesoid X Receptor (FXR) ELISA Kit

Principle of the Assay

The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Rat FXR. Standards or samples are added to the appropriate microtiter plate wells then with a biotin-conjugated antibody specific to Rat FXR. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Rat FXR, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of Rat FXR in the samples is then determined by comparing the OD of the samples to the standard curve.

For use with: Tissue homogenates, cell lysates, and other biological fluids.

For Research Use Only. Not for use in diagnostic procedures.

Target Information

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 pro-inflammatory (but not antiapoptotic) NF-kappa-B signaling.

GENE ID	60351
SWISS PROT	Q62735
SYNONYMS	NR1H4; BAR; FXR; HRR1; RIP14; Nuclear Receptor Subfamily 1,Group H,Member 4; Bile acid receptor; Farnesol receptor HRR-1; Retinoid X receptor-interacting protein 14

Materials Supplied

Kit Components	96 Wells Quantity/Size
Pre-Coated Microplate	48T: 6 strips x 8 wells; 96T: 12 strips x 8 wells
Standard (Lyophilized)	48T: 1 vial; 96T: 2 vials
Biotinylated Antibody (100×)	48T: 60 μL; 96T: 120 μL
Streptavidin-HRP (100×)	48T: 60 μL; 96T: 120 μL
Standard/Sample Diluent Buffer	48T: 10 mL; 96T: 20 mL
Biotinylated Antibody Diluent	48T: 6 mL; 96T: 12 mL
HRP Diluent	48T: 6 mL; 96T: 12 mL
Wash Buffer (25×)	48T: 10 mL; 96T: 20 mL
TMB Substrate Solution	48T: 6 mL; 96T: 10 mL
Stop Reagent	48T: 3 mL; 96T: 6 mL
Plate Covers	48T: 1 piece; 96T: 2 pieces

Storage

Storage

*If the kit is opened, Store the whole kit at 4°C. If the kit is not used up in 1 week. Store the Pre-Coated Microplate, Standard ,Biotinylated Antibody and Streptavidin-HRP at -20°C, the rest reagents at 4°C, please used up within 6 months. *If the kit is not opened, store the whole kit: 4°C(short time storage, valid for 6 months); -20°C (long-term storage, valid for 1 year). Avoid repeated freeze-thaw cycles.

Performance Characteristics

REPEATABILITY	Intra-assay Precision (Precision within an assay)：CV% < 8% Three samples of known concentration were tested twenty times on 1 plate to assess intra-assay precision. Inter-assay Precision (precision between assays)：CV% < 10% Three samples of known concentration were tested in forty separate assays to assess inter-assay precision.
SENSITIVITY	The minimum detectable dose was 0.051ng/mL.
ASSAY RANGE	0.16–10ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for detection of Rat FXR. No significant cross-reactivity or interference between Rat FXR and analogues was observed.