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Applications: Useful for studying protein binding and screening small molecules for drug discovery.
Assay Conditions: 50 µL reaction mix (50 mM HEPES pH 7.4, 50 mM NaCl, 200 µM CaCl2, 0.05% Tween-20, 0.1% BSA, and 0-1500 ng biotin-labeled PCSK9) is added to wells precoated with LDLR Ectodomain. Incubate for 2 hr. Add streptavidin-labeled HRP and incubate 1 hour. Finally, add HRP chemiluminescent substrates and read luminescence.
Background: PCSK9 is a crucial player in the regulation of plasma cholesterol homeostasis. It binds to low-density lipid receptor family members: low density lipoprotein receptor (LDLR), very low density lipoprotein receptor (VLDLR), apolipoprotein E receptor (LRP1/APOER) and apolipoprotein receptor 2 (LRP8/APOER2), and promotes their degradation. Acts via a non-proteolytic mechanism to enhance the degradation of the hepatic LDLR through a clathrin LDLRAP1/ARH-mediated pathway. May prevent the recycling of LDLR from endosomes to the cell surface or direct it to lysosomes for degradation. Can induce ubiquitination of LDLR leading to its subsequent degradation. Inhibits intracellular degradation of APOB via the autophagosome/lysosome pathway in a LDLR-independent manner. Involved in the disposal of non-acetylated intermediates of BACE1 in the early secretory pathway. Inhibits epithelial Na(+) channel (ENaC)-mediated Na(+) absorption by reducing ENaC surface expression primarily by increasing its proteasomal degradation. Regulates neuronal apoptosis via modulation of LRP8/APOER2 levels and related anti-apoptotic signaling pathways.
Description: Human proprotein convertase subtilisin/kexin type 9 (PCSK9), also known as FH3, HCHOLA3, and PC9, GenBank Accession No. NM_174936, a.a. 31-692(end), with C-terminal His and Avi tags, MW=73.8 kDa (calculated), expressed in an HEK293 cell expression system and enzymatically biotinylated using Avitag™ technology.
Format: Aqueous buffer solution
Formulation: 8 mM phosphate, pH 7.4, 110 mM NaCl, 2.2 mM KCl, and 20% glycerol.
Genbank: NM_174936
Storage Stability: At least 6 months at -80°C.
Tags: C-terminal His, Avi-Tags
Uniprot: Q8NBP7
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
References: 1. Abifadel, M., et al., Nat Genet. 2003 Jun,34(2):154-6.
2. Abifadel, M., et al., Hum Mutat. 2009 Apr;30(4):520-9.
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