Human PCSK9 coding sequence was cloned into the pcDNA3.1/V5-His TOPO vector (Invitrogen) and transfected into HEK293 cells. His-tagged PCSK9 was purified from the medium of transfected HEK293 cells using the ProBond protein purification system (Life technology). Protein purity was examined by gel electrophoresis with Coomassie blue staining. The purified PCSK9 concentration was determined using a human PCSK9 ELISA kit purchased from R&D System. Hamsters were fed a NCD or a HFD for three weeks prior to the injection of purified human PCSK9 (5μg per hamster) through the retro-orbital plexus under anesthesia. Blood was collected before injection and at the indicated time points after injection. Hamsters were euthanized at the end of the experiment and liver samples were collected. The level of human PCSK9 in hamster serum was determined using human PCSK9 ELISA kit (R & D system).
Human pcsk9 elisa kit
Manufactured by R&D Systems
Sourced in United States, United Kingdom
The Human PCSK9 ELISA kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) levels in human serum, plasma, and cell culture supernatants. The kit utilizes a specific antibody coated on a 96-well plate to capture PCSK9, and a detection antibody to quantify the captured PCSK9 using a colorimetric readout.
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3 protocols using human pcsk9 elisa kit
1
Purification and in vivo delivery of PCSK9
2
Quantifying ApoB and PCSK9 Secretion
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To detect the secreted amount of ApoB and PCSK9, the Human ApoB ELISA kit (Fine Test, Wuhan, China, cod. EH0620) and human PCSK9 ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA, 614 McKinley Place NE, cod. DY3888) were used according to the manufacturer’s instructions. Cells were seeded in 6 well plates (200,000 cells/well) in MEM/10% FBS, and 24 h later the treatments were performed. After 48 h the supernatant was collected, centrifuged at 15,000 rpm for 10 min and diluted according to the manufacturer’s instructions. Absorbance at 450 nm was obtained with VICTOR Nivo Multimode Microplate Reader (PerkinElmer, Waltham, MA, USA).
3
Quantitative PCSK9 Measurement by ELISA
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PCSK9 concentrations were measured by an enzyme-linked immunosorbent assay (Human PCSK9 ELISA Kit, R&D Systems, Cambridge, UK) using a spectrophotometric method at 450 nm. This assay employs a quantitative sandwich enzyme immunoassay technique using a monoclonal antibody specific for human PCSK9 that has been pre-coated onto a microplate. The PCSK9 from standards and samples are bound by the immobilized antibody and a second polyclonal antibody specific for human PCSK9 is then added to the wells. The CV of the reaction is less than 7%. According to the manufacturer, the normal range established in healthy volunteers is from 177 to 460 ng/mL.
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