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Product Catalog |
CircuLex PCSK9-LDLRCode No: CY-8150
Target:
PCSK9-LDLR Background:
PCSK9 (also known as neural apoptosis-regulated convertase, NARC-1) is a 692-residue extracellular protein expressed primarily in the kidneys, liver and intestines (1) representing the 9th member of the secretory subtilase family. Various genetic observations subsequently mapped PCSK9 as the third gene (along with LDLR and APOB) to cause autosomal dominant hypercholesterolemia (ADH). These studies suggested that gain of function mutations increase plasma levels of LDL-c (2–6), whereas nonsense or missense (loss-of-function) mutations, which interfere with folding or secretion of PCSK9, lead to a reduction of plasma levels of LDL-c and an 88% decrease in the risk of coronary heart disease (CHD) (5). In mice, adenoviral overexpression of PCSK9 results in increased plasma LDL-c level in normal mice but not in LDLR-deficient mice (7). Deletion of PCSK9 causes an increase in level of LDLR protein but not mRNA (8). These findings lead to a hypothesis that PCSK9 exerts its role in cholesterol metabolism through posttranslational down-regulation of LDLR, the receptor responsible for clearing LDL-c from plasma. Evidence is consistent with the secreted form of PCSK9 binding directly to the LDLR and resulting in degradation of the receptor (9, 10). Zhang et al. (11) localized the binding site of PCSK9 in the LDLR to the first epidermal growth factor-like repeat (EGF-A) of the extracellular domain and showed that PCSK9 binding to this site is required for LDLR degradation. In light of these observations and the fact that PCSK9 in the circulation may cause the degradation of hepatic LDLR in the liver, PCSK9 would seem to be an attractive drug target for lowering LDLC.
Size:
96 assays
Product Type:
Kit Kit Content:
All samples should be assayed in duplicate. The following components are supplied and are sufficient
for the one 96-well microtiter plate kit.
①Microplate: One microplate supplied ready to use, with 96 wells (12 strIPPs of 8-wells) in a foil,
zIPP-lock bag with a desiccant pack. Wells are coated with Recombinant LDL-EGF-AB domain.
②Recombinant His-tagged PCSK9 wild type: Lyophilized Recombinant His-tagged PCSK9 wild type.
Reconstitute contents of vial with 1.2 mL of H2O. Mix gently until dissolved. Final concentration of
recombinant His-tagged PCSK9 wild type solution should be 1 μg/mL. Unused 1 μg/mL Recombinant
His-tagged PCSK9 wild type solution can be stored in small aliquots (e.g. 200 μL) at below -70°C to
avoid repeated freezing and thawing.
③150X Biotinylated anti-His-tag monoclonal antibody: One vial containing 100 μL of 150X
Biotinylated anti-His-tag monoclonal antibody. For long term storage, unused 150X Biotinylated
anti-His-tag monoclonal antibody solution can be stored in small aliquots (e.g. 20 μL) at below -70°C to
avoid repeated freezing and thawing.
④Reaction Buffer: One bottle containing 60 mL of 1X buffer; used for binding reaction and dilution of
Recombinant His-tagged PCSK9 wild type. Ready to use.
⑤150X HRP-conjugated Streptavidin: One vial containing 100 μL of 150X HRP-conjugated
Streptavidin. For long term storage, unused 150X HRP-conjugated Streptavidin solution can be stored in
small aliquots (e.g. 20 μL) at below -70°C to avoid repeated freezing and thawing.
⑥Conjugate Dilution Buffer: One bottle containing 12 mL of 1X buffer; used for dilution of
HRP-conjugated Streptavidin. Ready to use
• 10X Wash Buffer: One bottle containing 100 mL of 10X Wash buffer.
• Substrate Reagent: One bottle containing 20 mL of the chromogenic substrate, tetra-methylbenzidine
(TMB). Ready to use.
• Stop Solution: One bottle containing 20 mL of 1 N H2SO4. Ready to use.
Storage Temp. (°C):
4
Shipping Temp. (°C):
4 References:
1. Seidah NG, Benjannet S, Wickham L, Marcinkiewicz J, Jasmin SB, Stifani S, Basak A, Prat A, Chretien M (2003) Proc Natl Acad Sci USA 100:928–933. 2. Abifadel M, Varret M, Rabes JP, Allard D, Ouguerram K, Devillers M, Cruaud C, Benjannet S, Wickham L, Erlich D, et al. (2003) Nat Genet 34:154–156. 3. Leren TP (2004) Clin Genet 65:419–422. 4. Allard D, Amsellem S, Abifadel M, Trillard M, Devillers M, Luc G, Krempf M, Reznik Y, Girardet JP, Fredenrich A, et al. (2005) Hum Mutat 26:497. 5. Cohen JC, Boerwinkle E, Mosley TH, Jr, Hobbs HH (2006) N Engl J Med 354, 1264–1272. 6. Berge KE, Ose L, Leren TP (2006) Arterioscler Thromb Vasc Biol 26:1094–1100. 7. Maxwell KN, Breslow JL (2004) Proc Natl Acad Sci USA 101:7100–7105. 8. Rashid S, Curtis DE, Garuti R, Anderson NN, Bashmakov Y, Ho YK, Hammer RE, Moon YA, Horton JD (2005) Proc Natl Acad Sci USA 102:5374–5379. 9. Lagace, T. A., Curtis, D. E., Garuti, R., McNutt, M. C., Park, S. W., Prather, H. B., Anderson, N. N., Ho, Y. K., Hammer, R. E., and Horton, J. D. (2006) J. Clin. Investig. 116, 2995–3005 10. Cameron, J., Holla, O. L., Ranheim, T., Kulseth, M. A., Berge, K. E., and Leren, T. P. (2006) Hum. Mol. Genet. 15, 1551–1558 11. Zhang, D. W., Lagace, T. A., Garuti, R., Zhao, Z., McDonald, M., Horton, J. D., Cohen, J. C., and Hobbs, H. H. (2007) J. Biol. Chem. 282, 18602–18612 Precautions:
• Allow all the components to come to room temperature before use. • All microplate strIPPs that are not immediately required should be returned to the zIPP-lock pouch, which must be carefully resealed to avoid moisture absorption. • Do not use kit components beyond the indicated kit expiration date. • Use only the microtiter wells provided with the kit. • Rinse all detergent residue from glassware. • Use deionized water of the highest quality. • Do not mix reagents from different kits. • The buffers and reagents used in this kit contain either sodium Kathon-CG as preservatives. Care should be taken to avoid direct contact with these reagents. • Do not mouth pIPPette or ingest any of the reagents. • Do not smoke, eat, or drink when performing the assay or in areas where samples or reagents are handled. • Dispose of tetra-methylbenzidine (TMB) containing solutions in compliance with local regulations. •CAUTION: Sulfuric Acid is a strong acid. Wear disposable gloves and eye protection when handling Stop Solution.
Intended Use:
For Research use only. Not for use in diagnostic procedure. |
