Recombinant Human APP/Protease Nexin II Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Inhibition Activity
Format
Carrier-Free

Order Details

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 #
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

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
Publications
Read Publications using
3466-PI in the following applications:

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.

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Publications for APP (3466-PI)(2)

We have publications tested in 2 confirmed species: Human, N/A.

We have publications tested in 2 applications: Enzyme Assay, Enzyme Substrate.


Filter By Application
Enzyme Assay
(1)
Enzyme Substrate
(1)
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(1)
N/A
(1)
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Lysosomal Dysfunction is Linked to Exosomal Secretion
By Christina Towers, PhD. Lysosomal Dysfunction and DiseaseLysosomes are highly acidic organelles that are critical for cellular function and indispensable for degradative pathways like autophagy and endocytosis....  Read full blog post.

Immunity’s flipside: Microglia promote Alzheimer’s pathology during inflammation
By Jamshed Arslan Pharm.D. Microglia are brain's macrophages. In Alzheimer's disease (AD), microglia clear up protein aggregates called amyloid beta plaques. The connection between immune activation and AD is unclea...  Read full blog post.

The C99 fragment of amyloid precursor protein (APP)
Alzheimer’s Disease (AD) is a neurodegenerative disorder that is characterized by an abundance of the beta-amyloid peptide in the brain.  When AD was first discovered, it was determined that beta-amyloid was produced as a result of the prote...  Read full blog post.

Beta Amyloid (MOAB2) and the link between traumatic brain injury and Alzheimer’s disease
An epidemiological association between traumatic brain injury (TBI) and Alzheimer's disease (AD) has long been established.  Interestingly, an increase in beta amyloid  (one hallmark of AD) directly following TBI has been observed.  In fact, it h...  Read full blog post.

Niemann Pick-C1 and cholesterol dynamics
Niemann-Pick type C1 (NPC1) mediates low-density cholesterol transport from late endosomes and lysosomes to other areas of the cell via receptor mediation endocytosis.  Although cholesterol moves freely inside the cell, it cannot independently expo...  Read full blog post.

FANCD2 and DNA damage repair
Fanconi anemia (FA) is a genetically inherited disorder that yields cytogenetic instability, hypersensitivity to DNA crosslinking compounds and defective DNA repair. A variety of genes have been identified within the FA pathway that are referred t...  Read full blog post.

Beta Amyloid Neurotoxicity and Alzheimer's Disease
A major histopathological hallmark of Alzheimer's disease (AD) is the presence of amyloid deposits in the parenchyma of the amygdala, hippocampus, and neocortex. The principal component of amyloid is beta amyloid (AB). The pathologic accumulation of A...  Read full blog post.

ApoE: The Key to Preventing Alzheimer's Disease?
Apolipoprotein E also known as ApoE is a 36kDa protein that is expressed in all lipoprotein fractions in plasma. This protein is produced in high quantities in the liver, brain, spleen, lung and kidney. The function of APOE is to mediate the binding,...  Read full blog post.

Amyloid beta and Methionine Sulfoxide Related to Abeta 42 Antibody and Abeta 40 Antibody
By Eric NeeleyAlzheimer's disease is a devastating neurodegenerative illness characterized by the formation of plaques, tangles, and eventually synaptic loss. Amyloid beta (A beta ) is the processed form of the Amyloid precursor protein (APP), and whose...  Read full blog post.

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Bioinformatics

Gene Symbol APP
Entrez
Uniprot