PCSK9 Protein, Human (HEK293, V474I, G670E, His)

The PCSK9 protein is an important regulator of plasma cholesterol homeostasis, affecting LDL receptor family members such as LDLR and VLDLR. It promotes their intracellular degradation and enhances hepatic LDLR degradation through non-proteolytic mechanisms. PCSK9 Protein, Human (HEK293, V474I, G670E, His) is the recombinant human-derived PCSK9 protein, expressed by HEK293 , with C-6*His labeled tag and V474I, G670E mutation.

PCSK9 protein emerges as a pivotal regulator in the intricate orchestration of plasma cholesterol homeostasis. Demonstrating its influence on low-density lipid receptor family members, including the low-density lipoprotein receptor (LDLR), very low-density lipoprotein receptor (VLDLR), apolipoprotein E receptor (LRP1/APOER), and apolipoprotein receptor 2 (LRP8/APOER2), PCSK9 facilitates their degradation within intracellular acidic compartments. Employing a non-proteolytic mechanism, it enhances the hepatic LDLR degradation through a clathrin LDLRAP1/ARH-mediated pathway, possibly impeding LDLR recycling and directing it toward lysosomal degradation. Moreover, PCSK9 exhibits LDLR-independent inhibition of APOB intracellular degradation via the autophagosome/lysosome pathway and plays a role in the disposal of non-acetylated BACE1 intermediates in the early secretory pathway. Notably, it regulates epithelial Na(+) channel (ENaC)-mediated Na(+) absorption by augmenting ENaC proteasomal degradation, and influences neuronal apoptosis through the modulation of LRP8/APOER2 levels and associated anti-apoptotic signaling pathways.

1.The enzyme activity of this recombinant protein is testing in progress, we cannot offer a guarantee yet.
2.Measured by its binding ability in a functional ELISA. When Recombinant Human LDLR is coated at 2 µg/mL can bind Recombinant Human PCSK9. The ED₅₀ for this effect is 188.6 ng/mL.

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  Measured by its binding ability in a functional ELISA. When Recombinant Human LDLR is coated at 2 µg/mL can bind Recombinant Human PCSK9. The ED₅₀ for this effect is 188.6 ng/mL.

Materials
PCSK9 Protein, Human (HEK293, V474I, G670E, His) （HY-P70545）
Biotin
Pre-cooled Sterile Water
Washing Buffer: 1×PBST
Blocking Buffer: PBST with 1% BSA
TMB Chromogenic Solution: Mix Chromogenic Solution A and Chromogenic Solution B at a ratio of 1:1 before use; prepare freshly as needed
Stop Solution: 2 M H₂SO₄

Procedure
Biotin Labeling
1. Dissolution of Biotin reagent: Remove Biotin from -10~-30°C, immediately dissolve 1 tube of Biotin with 180 μL of cold pure water. Gently pipette to mix, preparing 10 mM Biotin.
2. Calculate the volume of EZ-Link^TM Sulfo-NHS-LC-LC-Biotin, No-Weigh^TM Format to be added.
3. Incubate at room temperature in the dark for 45 min.
4. Quantification of labeled protein: Perform protein concentration quantification using BCA assay. The concentration of the labeled Biotinylated Human PCSK9 is 1670 μg/mL.

ELISA Detection
1. Coating: Dilute Recombinant Human LDLR protein to 2 μg/mL with 1×PBS, add 100 μL/well, and incubate at 4°C overnight.
2. Plate washing: Wash the plate with washing buffer, 260 μL/well, once, then pat dry.
3. Blocking: Use blocking buffer (1×PBST containing 1% BSA), 150 μL/well, block at 25°C with 450 rpm for 4 h.
4. Dilute Biotinylated Human PCSK9 to the following concentrations: 0, 0.25, 0.5, 2.5, 5, 25, 50, 250, 500, 2500, 5000, 25000, 50000 ng/mL.
5. Add Biotinylated Human PCSK9: 100 μL/well, incubate at 25°C with 450 rpm for 2 h.
6. Plate washing: Wash the plate with washing buffer, 260 μL per well, 5 times, with 1 min retention each time, then pat dry.
7. Add secondary antibody: Dilute the secondary antibody at a ratio of 1:2000, 100 μL/well, incubate at 25°C with 450 rpm for 1 h.
8. Plate washing: Wash the plate with washing buffer, 260 μL/well, 3 times, with 1 min retention each time, then pat dry.
9. Add TMB chromogenic solution: 100 μL/well; incubate at room temperature in the dark for 15 min.
10. Terminate the reaction: Add 2 M H₂SO₄, 100 μL/well.
11. Plate reading: Read at 450 nm.

Human

HEK293

C-6*His

Q8NBP7 (Q31-Q692, V474I, G670E)

255738  [NCBI]

Full Length of Mature Protein (with Propeptide)

QEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQSIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGASMRSLRVLNCQGKGTVSGTLIGLEFIRKSQLVQPVGPLVVLLPLAGGYSRVLNAACQRLARAGVVLVTAAGNFRDDACLYSPASAPEVITVGATNAQDQPVTLGTLGTNFGRCVDLFAPGEDIIGASSDCSTCFVSQSGTSQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFPEDQRVLTPNLVAALPPSTHGAGWQLFCRTVWSAHSGPTRMATAIARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSEEAVTAVAICCRSRHLAQASQELQ

Approximately 19(Propeptide)&60 (Mature Protein) kDa, based on SDS-PAGE under reducing conditions, due to the glycosylation.

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  Greater than 95% as determined by reducing SDS-PAGE.

Solution.

Supplied as a 0.22 μm filtered solution of 20 mM NaH₂PO₄, 150 mM NaCl, 0.1 M Arginine, 0.1 M Glu, 0.01% Tween 20, pH 7.4.

<1 EU/μg, determined by LAL method.

N/A.

Stored at -80°C for 1 year from date of receipt. It is stable at -20°C for 3 months after opening. It is recommended to freeze aliquots at -80°C for extended storage. Avoid repeated freeze-thaw cycles.

Shipping with dry ice.