Recombinant Human Neprilysin (carrier-free)

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Other Names
CD10, MME, NEP, SFE, CALLA, CMT2T, SCA43, Membrane Metalloendopeptidase
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Human_Neprilysin_RECOM_CF_1_051920
Human Neprilysin activity is measured by its ability to cleave the fluorogenic peptide substrate MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH. The specific activity is > 1500 pmol/min/µg in the presence of 0.005 µg of recombinant human Neprilysin.
  • Human_Neprilysin_RECOM_CF_1_051920
    Human Neprilysin activity is measured by its ability to cleave the fluorogenic peptide substrate MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH. The specific activity is > 1500 pmol/min/µg in the presence of 0.005 µg of recombinant human Neprilysin.
  • Human_Neprilysin_RECOM_CF_2_051920
    Stability Testing for Recombinant Human Neprilysin. Recombinant human Neprilysin was aliquoted in Tris pH 7.2, 50 mM NaCl, 5% glycerol at 0.2 mg/mL. One aliquot was frozen and thawed four times (4x Freeze/Thaw) and compared to the control that was kept at 4°C (Control). The samples were tested for their ability to cleave the fluorogenic peptide substrate MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH. The specific activity is > 1500 pmol/min/µg.
Cat # Size Price Save
790502 10 µg ¥13,500
790504 25 µg ¥22,500
790506 100 µg ¥51,300
790508 500 µg ¥153,000
Description

Neprilysin is a type II transmembrane glycoprotein zinc-dependent metalloproteinase with broad expression in the body. It is a neutral endopeptidase and cleaves peptides on amino sides of hydrophobic residues. Neprilysin has activity in vasoactive peptides (adrenomodullin, angiotensin I, angiotensin II), natriurietic peptides (ANP, BNP, CNP, urodilatin), bradikynin, kallidin, endothelin, neurokinin A, neuropeptide Y, and substance P. Also, it has activity in amyloid β, galanin, bombesin-like peptides, adrenocorticotrophic hormone, gonadotropin-releasing hormone, α-melanocyte stimulating hormone, and oxytocyne. In addition, it has activity in peptides associated with digestion and metabolism (cholecystokinin, gastrin-releasing peptide, glucagon, and glucagon-like peptides). The recombinant protein is an antigen for common acute lymphocitic leukemia. Decrease of neprilysin has been associated with early stages of Alzheimer's disease, since neprilysin has been identified as a main physiological amyloid β peptide-degrading enzyme. Neprilysin is also known as acute lymphocytic leukemia antigen, and it is expressed in normal precursor B cells (in bone marrow) and normal germinal centers B cells.

Product Details
Technical data sheet

Product Details

Source
Human, amino acid (Tyr52-Trp750) (Accession: NM_000789.4), with a N-terminal 6His-GGS tag was expressed in 293E cell.
Molecular Mass
The 708 amino acid recombinant protein has a predicted molecular mass of approximately 80.8 kD. The DTT-reduced and non-reduced protein migrates at approximately 80 kD by SDS-PAGE. The predicted N-terminal amino acid is His.
Purity
> 95%, as determined by Coomassie stained SDS-PAGE
Formulation
Tris pH 7.2, 50 mM NaCl, 5% glycerol
Concentration
10-25 µg sizes are bottled at 200 µg/mL. 100 µg and larger sizes are lot-specific and bottled at the concentration indicated on the vial. (Please contact technical support for concentration, or use our Lookup tool if you have a lot number.)
Storage & Handling
Unopened vial can be stored at -20°C or -70°C for six months. For maximum results, quick spin vial prior to opening. Avoid repeated freeze/thaw cycles.
Activity
Human Neprilysin activity is measured by its ability to cleave the fluorogenic peptide substrate MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH. The specific activity is > 1500 pmol/min/µg in the presence of 0.005 µg of recombinant human Neprilysin.
Application

Bioassay

Application Notes

Human Neprilysin Enzymatic Assay

Human Neprilysin (CD10) activity is measured by its ability to cleave the fluorogenic peptide substrate MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH. The increase of the product is monitored by an increase in intensity of fluorescence at 405 nm with excitation at 320 nm. Perform the assay rapidly. The specific activity is > 1500 pmol/min/µg in the presence of 0.005 µg of recombinant human Neprilysin.


Materials

  • Assay Buffer: 50 mM Tris, 0.05% Brij-35, pH 9.0
  • Human Neprilysin
  • Fluorogenic peptide substrate: MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH
  • F16 Black Maxisorp Plate (Nunc, Cat. No. 475515)


Activity Assay Procedure

  1. Dilute the substrate in assay buffer at 20 µM. Wrap the substrate container in aluminium foil.
  2. In assay buffer, prepare 50 µL of serial dilutions of human Neprilysin. Start at a concentration of 3.2 µg/mL and dilute 1 to 2 ten times after.
  3. Add 50 µL of 20 µM substrate to wells with 50 µL of prepared dilutions.
  4. Include a substrate blank with 50 µL of assay buffer and 50 µL of 20 µM substrate without any human Neprilysin.
  5. Read at excitation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes.
  6. Calculate specific activity using the following formula:

Specific Activity (pmol/min/µg)=  ((Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU))
                                                                                                  amount of enzyme (µg)

 


Final assay conditions per well:
Human Neprilysin: 0.16, 0.08, 0.04, 0.02, 0.01, 0.005, 0.0025, 0.00125, 0.000625, 0.0003125, 0.00015625 µg.
Substrate: 10 µM

BioLegend carrier-free recombinant proteins provided in liquid format are shipped on blue ice. Our comparison testing data indicates that when handled and stored as recommended, the liquid format has equal or better stability and shelf-life compared to commercially available lyophilized proteins after reconstitution. Our liquid proteins are verified in-house to maintain activity after shipping on blue ice and are backed by our 100% satisfaction guarantee. If you have any concerns, contact us at tech@biolegend.com.

Antigen Details

Structure
Monomer
Distribution

Leukemic cells, pre B cells, germinal centers B cells, granulocytes, and fibroblasts. Abundant expression in duodenum, kidney, fat, and small intestine.  

Function
Inactivates peptide hormones such as bradykinin, substance P, neurotensin, enkephalins, and glucagon.
Interaction
Bradykinin, natriuretic peptides, and adrenomedullin, glucagon, enkephalins, substance P, neurotensin, angiotensin-1, angiotensin-2, and oxytocin.
Bioactivity
Human Neprilysin cleaves a fluorogenic peptide substrate MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH. The specific activity is > 1500 pmol/min/µg in the presence of 0.005 µg of recombinant human Neprilysin.
Cell Sources
293E cell
Cell Type
B cells, Endothelial cells, Epithelial cells, Fibroblasts, Leukemia
Biology Area
Cancer Biomarkers, Cardiovascular Biology, Cell Biology, Inhibitory Molecules, Innate Immunity, Neuroscience
Molecular Family
Enzymes and Regulators, Proteases
Antigen References
  1. N Iwata, et al., 2001. Science. 292(5521):1550.
  2. S Zraika, et al., 2013. Diabetes. 62:1593.
  3. C Bavishi, et al. 2015. Eur. Heart J. 36:1967.
  4. E Riddell, and JM Vader. 2017. Curr. Heart Fail. Rep. 14:134.
  5. DJ Campbell. 2018. Front Med. 5:257.
Gene ID
4311 View all products for this Gene ID
UniProt
View information about Neprilysin on UniProt.org

Related FAQs

Why choose BioLegend recombinant proteins?

     • Each lot of product is quality-tested for bioactivity as indicated on the data sheet.
     • Greater than 95% Purity or higher, tested on every lot of product.
     • 100% Satisfaction Guarantee for quality performance, stability, and consistency.
     • Ready-to-use liquid format saves time and reduces challenges associated with reconstitution.
     • Bulk and customization available. Contact us.
     • Learn more about our Recombinant Proteins.

How does the activity of your recombinant proteins compare to competitors?

We quality control each and every lot of recombinant protein. Not only do we check its bioactivity, but we also compare it against other commercially available recombinant proteins. We make sure each recombinant protein’s activity is at least as good as or better than the competition’s. In order to provide you with the best possible product, we ensure that our testing process is rigorous and thorough. If you’re curious and eager to make the switch to BioLegend recombinants, contact your sales representative today!

What is the specific activity or ED50 of my recombinant protein?

The specific activity range of the protein is indicated on the product datasheets. Because the exact activity values on a per unit basis can largely fluctuate depending on a number of factors, including the nature of the assay, cell density, age of cells/passage number, culture media used, and end user technique, the specific activity is best defined as a range and we guarantee the specific activity of all our lots will be within the range indicated on the datasheet. Please note this only applies to recombinants labeled for use in bioassays. ELISA standard recombinant proteins are not recommended for bioassay usage as they are not tested for these applications.

Have your recombinants been tested for stability?

Our testing shows that the recombinant proteins are able to withstand room temperature for a week without losing activity. In addition the recombinant proteins were also found to withstand four cycles of freeze and thaw without losing activity.

Does specific activity of a recombinant protein vary between lots?

Specific activity will vary for each lot and for the type of experiment that is done to validate it, but all passed lots will have activity within the established ED50 range for the product and we guarantee that our products will have lot-to-lot consistency. Please conduct an experiment-specific validation to find the optimal ED50 for your system.

How do you convert activity as an ED50 in ng/ml to a specific activity in Units/mg?
Use formula Specific activity (Units/mg) = 10e6/ ED50 (ng/mL)
Go To Top Version: 1    Revision Date: 05/19/2020

For research use only. Not for diagnostic use. Not for resale. BioLegend will not be held responsible for patent infringement or other violations that may occur with the use of our products.

 

*These products may be covered by one or more Limited Use Label Licenses (see the BioLegend Catalog or our website, www.biolegend.com/ordering#license). BioLegend products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products, reverse engineer functionally similar materials, or to provide a service to third parties without written approval of BioLegend. By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. Unless otherwise indicated, these products are for research use only and are not intended for human or animal diagnostic, therapeutic or commercial use.

 

BioLegend Inc., 8999 BioLegend Way, San Diego, CA 92121 www.biolegend.com
Toll-Free Phone: 1-877-Bio-Legend (246-5343) Phone: (858) 768-5800 Fax: (877) 455-9587

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