Recombinant Human APP/Protease Nexin II Protein, CF

• Reactivity: Hu
• Applications: Inhibition Activity
• Format: Carrier-Free

Recombinant Human APP/Protease Nexin II Protein, CF Summary

• Details of Functionality: Measured by its ability to inhibit trypsin cleavage of a fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH₂ (Catalog # ES002). The IC₅₀ value is <1.2 nM, as measured with under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human APP protein
  Leu18-Leu688, with a C-terminal 6-His tag
• Accession #: P05067
• N-terminal Sequence: Leu18
• Protein/Peptide Type: Recombinant Enzymes
• Gene: APP
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Inhibition Activity
• Theoretical MW: 77 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 120 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Reconstitution Instructions: Reconstitute at 200 μg/mL in sterile, deionized water.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35, pH 7.5 (TCNB)
  • Recombinant Human APP/Protease Nexin II (rhAPP) (Catalog # 3466-PI)
  • Trypsin (Sigma, Catalog # T-1426)
  • Fluorogenic peptide substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute Trypsin to 0.400 μg/mL in Assay Buffer.
  2. Prepare a curve of rhAPP-770 (MW: 76,875 Da) in Assay Buffer. Make the following serial dilutions: 100, 20, 10, 5, 3.33, 2.22, 1.11 and 0.370 nM.
  3. For each reaction tube mix 20 μL of the diluted Trypsin, 100 μL of the rhAPP curve, and 80 μL of TCNB for a final volume of 200 μL. Include a control (in duplicate) containing 180 μL of Assay Buffer and 20 μL of the diluted Trypsin.
  4. Incubate mixtures at room temperature for 30 minutes.
  5. Dilute substrate to 20 μM in Assay Buffer.
  6. Load in a black well plate 50 μL of the reactions, and start the reaction by adding 50 μL of 20 μM substrate.
     Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  7. Derive the 50% inhibiting concentration (IC₅₀) value for rhAPP by plotting RFU/min (or specific activity) vs. concentration with 4‑PL fitting.
  8. The specific activity for Trypsin at each point may be determined using the following formula (if needed):

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

  *Adjusted for Substrate Blank
  **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

  Per Well:
  • Trypsin: 0.002 μg
  • rhAPP curve: 25, 5, 2.5, 1.25, 0.833, 0.555, 0.2775 and 0.0925 nM
  • Substrate: 10 μM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human APP/Protease Nexin II Protein, CF

• amyloid beta (A4) precursor protein-binding, family B, member 2
• amyloid beta A4 precursor protein-binding family B member 2
• Amyloid beta precursor protein
• Amyloid beta
• APP
• beta Amyloid
• Protease Nexin II

Background

Amyloid precursor protein (APP) is a type I membrane protein with several human isoforms due to alternative splicing. APP-770, -751, and -733 contain a Kunitz protease inhibitor (KPI) domain (residue 291-342) and APP-695 does not. APP is a cell surface molecule with many functions. It can be processed proteolytically in two different pathways. In one pathway, beta - and gamma -secretase cleave at the  beta  site between residue 670 and 671 and the gamma site between residue 711 and 714 to produce beta ‑amyloid peptide (A beta 40 and A beta 42), a major component in plaques found in brains of patients with Alzheimer's disease (1). The other pathway involves alpha -secretase that cleaves residues between 687 and 688. It is antiamyloidogenic due to its benign character and the prevention of the A beta peptide formation (2). Soluble APP containing the KPI domain, also referred to as protease nexin II, is a potent inhibitor of serine proteases and may have additional functions. For example, it may regulate the contact face of blood coagulation and limit thrombosis specially in brain due to its localization and coagulation factor XI inhibiting activity (3, 4).

1. Haass, C. (2004) EMBO J. 23:483.
2. Lichtenthaler, S. F. and C. Haass (2004) J. Clin. Invest. 113:1384.
3. Badellino, K.O. and P.N. Walsh (2000) Biochemistry 39:4769.
4. Xu, F. et al. (2005) Proc. Natl. Acad. Sci USA. 102:18135.

Publications for APP (3466-PI)(2)

Publications using 3466-PI

• Thomas RS, Liddell JE, Kidd EJ Anti-amyloid precursor protein immunoglobulins inhibit amyloid-beta production by steric hindrance. FEBS J., 2010-12-01;278(1):167-78. 2010-12-01 [PMID: 21122073] (Enzyme Assay, Human)
• Ahmed RR, Holler CJ, Webb RL BACE1 and BACE2 enzymatic activities in Alzheimer&#039;s disease. J. Neurochem., 2009-12-04;112(4):1045-53. 2009-12-04 [PMID: 19968762] (Enzyme Assay Substrate)

Bioinformatics

• Gene Symbol: APP
• Uniprot:
  • P05067()