PCSK9 蛋白, 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 蛋白在血浆胆固醇稳态的复杂协调中发挥着关键调节作用。证明其对低密度脂蛋白受体家族成员的影响，包括低密度脂蛋白受体 (LDLR)、极低密度脂蛋白受体 (VLDLR)、载脂蛋白 E 受体 (LRP1/APOER) 和载脂蛋白受体 2 (LRP8/APOER2) ），PCSK9 促进它们在细胞内酸性区室中的降解。它采用非蛋白水解机制，通过网格蛋白 LDLRAP1/ARH 介导的途径增强肝脏 LDLR 降解，可能阻碍 LDLR 回收并将其引导至溶酶体降解。此外，PCSK9 通过自噬体/溶酶体途径表现出 LDLR 独立的 APOB 细胞内降解抑制作用，并在早期分泌途径中非乙酰化 BACE1 中间体的处理中发挥作用。值得注意的是，它通过增强 ENaC 蛋白酶体降解来调节上皮 Na(+) 通道 (ENaC) 介导的 Na(+) 吸收，并通过调节 LRP8/APOER2 水平和相关的抗凋亡信号通路影响神经元凋亡。

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.

• 
  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.

溶液.

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.