The Low-Density Lipoprotein (LDL) Receptor is a mosaic protein of 839aa (after removal of 21aa signal peptide) that mediates the endocytosis of cholesterol-rich LDL. In humans, the LDL receptor protein is encoded by the LDLR gene. It belongs to the Low density lipoprotein receptor gene family. The low density lipoprotein receptor (LDLR) gene family consists of cell surface proteins involved in receptor-mediated endocytosis of specific ligands. Low density lipoprotein (LDL) is normally bound at the cell membrane and taken into the cell ending up in lysosomes where the protein is degraded and the cholesterol is made available for repression of microsomal enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the rate-limiting step in cholesterol synthesis. At the same time, a reciprocal stimulation of cholesterol ester synthesis takes place. Mutations in this gene cause the autosomal dominant disorder, familial hypercholesterolemia. Alternate splicing results in multiple transcript variants. The LDLR BioAssay(TM) ELISA Kit is for the quantitative detection of human LDLR in cell lysates, cell culture supernates, serum, plasma (Heparin) and urine. Detection Range: 62.5-4000pg/ml Sensitivity: <12pg/ml Specificity: Recognizes natural and recombinant human LDLR. There is no detectable cross-reactivity with other relevant proteins. Test Principle: The LDLR BioAssay(TM) ELISA Kit is a Sandwich High Sensitivity ELISA is a solid phase immunoassay specially designed to measure human LDLR with a 96-well strip plate that is pre-coated with antibody specific for LDLR. The detection antibody is a biotinylated antibody specific for LDLR. The kit contains recombinant human LDLR with immunogen: Expression system for standard: NS0 cells, Immunogen sequence: A22-R788. To measure human LDLR, add standards and samples to the wells, then add the biotinylated detection antibody. Wash the wells with PBS buffer, and add Avidin-Biotin-Peroxidase Complex (ABC-HRP). Wash away the unbounded ABC-HRP with PBS buffer and add TMB. TMB is substrate to HRP and will be catalyzed to produce a blue color product, which changes into yellow after adding acidic stop solution. The density of the yellow product is linearly proportional to human LDLR in the sample. Read the density of the yellow product in each well using a plate reader, and benchmark the sample wells readings against the standard curve to determine the concentration of human LDLR in the sample. Kit Components: 396709A: Microtiter Strips, 1x96 wells *396709B: LDLR Standard, 2x10ng 396709C: Anti-human LDLR (Biotin), 1x100ul (1:100 dilution) 143696: Avidin-Biotin-Peroxidase Complex (ABC), 1x100ul (1:100 dilution) 143697: Sample Diluent Buffer, 1x30ml 143698: Antibody Diluent Buffer, 1x12ml 143699: ABC Diluent Buffer, 1x12ml 143700: TMB Color Developing Agent, 1x10ml 143701: TMB Stop Solution, 2N H2SO4, 1x10ml 143702: Wash Buffer, 25X, 1x20ml Storage and Stability: Store *396709B powder at 4C. Once reconstituted store at 4C for up to 12 hours or at -20C for up to 48 hours. Store other components at 4C. Stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Assay Summary: 1. Add 100ul of samples and standards and incubate the plate at 37C for 90 minutes or at RT for 2 hours. Do not wash. 2. Add 100ul biotinylated antibody and incubate the plate at 37C for 60 minutes or at RT for 90 minutes. Wash plate 3 times with Working Wash Buffer. 3. Add 100ul of ABC working solution and incubate the plate at 37C for 30 minutes or at RT for 40 minutes. Wash plate 5 times with Working Wash Buffer. 4. Add 90ul of TMB color developing agent and incubate the plate at 37C in dark for 15-25 minutes or at RT for 30 minutes. 5. Add 100ul TMB Stop Solution and read.
