ACE2 蛋白, Human (HEK293, mFc)

ACE2 Protein, Human (HEK293, mFc) is a SARS-CoV-2 receptor. Angiotensin-Converting Enzyme 2 efficiently hydrolyses the potent vasoconstrictor angiotensin II to angiotensin. It is a consequence of this action that ACE2 participates in the renin-angiotensin system.

血管紧张素转换酶 2 (ACE2) 是一种 I 型整合膜蛋白，充当羧肽酶，从其底物的 C 末端切割单个疏水/碱性残基。ACE2 有效地将强效血管收缩剂血管紧张素 II 水解为血管紧张素。ACE2 参与肾素-血管紧张素系统就是这种作用的结果。然而，ACE2 也会水解强啡肽 A (1–13)、apelin-13 和 des-Arg9 缓激肽。ACE2 在这些肽系统中的作用尚未被揭示。ACE2 的生理作用与高血压、心脏功能、心脏功能和糖尿病有关，并且是严重急性呼吸综合征冠状病毒的受体。本文回顾了 ACE2 的生物化学，并讨论了一些重要发现，例如 ACE2 和睾丸 ACE 晶体结构的阐明以及 ACE2 抑制剂的开发，这些发现为该酶的未来研究奠定了基础。

Measured by its binding ability in a functional ELISA. Immobilized Human ACE2, at 2 μg/mL (100 μL/well) can bind Biotinylated SARS-CoV-2 S1 protein. The ED₅₀ for this effect is 8.465 ng/mL, corresponding to a specific activity is 1.18×10^5 Unit/mg.

• 
  Measured by its binding ability in a functional ELISA. Immobilized Human ACE2,at 2 μg/mL (100 μL/well) can bind Biotinylated SARS-CoV-2 S1 protein. The ED₅₀ for this effect is 8.465 ng/mL, corresponding to a specific activity is 1.18×10⁵ Unit/mg.

Materials
1. ACE2 Protein, Human (HEK293, mFc) (HY-P7443)
2. Biotin
3. Pre-chilled Sterile Water
4. Washing buffer: 1×PBST
5. Blocking buffer: PBST with 1% BSA
6. TMB Chromogenic Solution: Mix Chromogenic Solution A and Chromogenic Solution B at a ratio of 1:1 before use, and prepare freshly as needed
7. Stop solution: 2 M H₂SO₄

Procedure
Biotin Labeling
1. Dissolution of Biotin reagent: Take out Biotin from -10~-30℃, immediately dissolve 1 tube of Biotin with 180 μL of cold pure water. Gently pipette to mix well to prepare 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: Use BCA assay for protein concentration quantification; the concentration of the labeled Biotinylated SARS-CoV-2 S1 is 208 μg/mL.

ELISA Detection
1. Coating: Dilute ACE2 protein to 2 μg/mL with 1×PBS, add 100 μL per well, and incubate overnight at 2-8℃.
2. Plate washing: Wash the plate with washing buffer, 260 μL per well, wash once, and pat dry.
3. Blocking: Use 1×PBST blocking buffer containing 1% BSA, add 150 μL per well, and block at 25℃ with 450 rpm shaking for 4 h.
4. Dilute Biotinylated SARS-CoV-2 S1 to the following concentrations: 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500, 1000, 2000 ng/mL.
5. Add receptor protein: 100 μL per well, incubate at 25℃ with 450 rpm shaking for 2 h.
6. Plate washing: Wash the plate with washing buffer, 260 μL per well, wash 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, add 100 μL per well, and incubate at 25℃ with 450 rpm shaking for 1 h.
8. Plate washing: Wash the plate with washing buffer, 260 μL per well, wash 3 times with 1 min retention each time, and pat dry.
9. Add TMB chromogenic solution: 100 μL per well, incubate at room temperature in the dark for 15 min.
10. Terminate the reaction: Add 2 M H₂SO₄, 100 μL per well.
11. Plate reading: Read the absorbance at 450 nm.
12. Use GraphPad Prism software to plot a curve with OD value as the ordinate and Biotinylated SARS-CoV-2 S1 concentration as the abscissa, and calculate the ED₅₀ value.

Human

HEK293

C-mFc

Q9BYF1-1 (Q18-S740)

59272  [NCBI]

Extracellular Domain

QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVS

110-140 kDa

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

溶液.

Supplied as a 0.22 μm filter solution of 20 mM Tris-HCl, 300 mM NaCl, 1 mM ZnCl₂, 10% Glycerol, pH 7.4.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<1 EU/μg, determined by LAL method.

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.

• [1]. Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target.

  [2]. Warner FJ, et al. Angiotensin-converting enzyme-2: a molecular and cellular perspective. Cell Mol Life Sci. 2004 Nov;61(21):2704-13.  [Content Brief]