Proprotein convertase subtilisin/kexin type 9, also known as PCSK9, is an enzyme that in humans is encoded by the PCSK9 gene. This gene encodes a proprotein convertase belonging to the proteinase K subfamily of the secretory subtilase family. By genomic sequence analysis, the PCSK9 gene was mapped to chromosome 1p32. This protein plays a major regulatory role in cholesterol homeostasis. PCSK9 binds to the epidermal growth factor-like repeat A(EGF-A) domain of the low-density lipoprotein receptor(LDLR), inducing LDLR degradation. Reduced LDLR levels result in decreased metabolism of low-density lipoproteins(LDL), which could lead to hypercholesterolemia. PCSK9 may also have a role in the differentiation of cortical neurons.
Intended Use:This kit is intended for the quantitative determination of human PCSK9 in cell culture supernatants, cell lysates, tissue homogenates, serum and plasma (heparin, EDTA).
Principle of the Assay:The Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9), BioAssay™ ELISA Kit (Human) is based on standard sandwich enzyme-linked immunosorbent assay technology. A mouse monoclonal antibody specific for PCSK9 has been pre-coated onto 96-well plates. Standards (E. coli-expressed recombinant fragment corresponding to S153 thru Q692 of human PCSK9) and test samples are added to the wells, incubated and then biotinylated detection goat polyclonal antibody specific for PCSK9 is added, followed by an incubation step and a washing step with PBS or TBS buffer. Avidin-Biotin-Peroxidase Complex is added and unbound conjugates are washed away with PBS or TBS buffer. HRP substrate TMB is used to visualize the HRP-mediated reaction. TMB is catalyzed by HRP to produce a blue colored product that changes into yellow upon addition of acidic stop solution. The intensity of the yellow color is proportional to the amount of human PCSK9 in the sample.
Storage and Stability:Unopened kit is stable at 4°C for 6 months.
Material Required But Not Provided:1. Microplate reader in standard size2. Automated plate washer3. Adjustable pipettes and pipette tips. Multichannel pipettes are recommended when a large number of samples are to be assayed 4. Clean tubes and Eppendorf tubes5. Washing buffer (neutral PBS or TBS)
Assay Summary:1. Add samples and standards and incubate the plate at 37°C for 90 min. Do not wash. 2. Add biotinylated antibody and incubate the plate at 37°C for 60 min. Wash plate 3 times with 0.01M TBS or PBS
3. Add ABC working solution and incubate the plate at 37°C for 30min. Wash plate 5 times with 0.01M TBS or PBS
4. Add TMB color developing agent and incubate the plate at 37°C in dark for 20-25 min
5. Add Stop solution and read absorbance at 450nm.
