Recombinant Human APP/Protease Nexin II Protein, CF

Catalog #: 3466-PI
2 Citations Datasheet / COA / SDS

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3466-PI-010

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Recombinant Human APP/Protease Nexin II Protein, CF Summary

Product Specifications

Purity

>90%, by SDS-PAGE under reducing conditions and visualized by silver stain

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

Activity

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
Analysis

Leu18

Predicted Molecular Mass

77 kDa

SDS-PAGE

120 kDa, reducing conditions

Product Datasheets

3466-PI

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

3466-PI

Formulation	Lyophilized from a 0.2 μm filtered solution in PBS.
Reconstitution	Reconstitute at 200 μg/mL in sterile, deionized water.
Shipping	The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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.

Assay Procedure

Materials

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

Dilute Trypsin to 0.400 μg/mL in Assay Buffer.
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.
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.
Incubate mixtures at room temperature for 30 minutes.
Dilute substrate to 20 μM in Assay Buffer.
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.
Derive the 50% inhibiting concentration (IC₅₀) value for rhAPP by plotting RFU/min (or specific activity) vs. concentration with 4‑PL fitting.
The specific activity for Trypsin at each point may be determined using the following formula (if needed):

Specific Activity (pmol/min/µg) =	Adjusted V_max* (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

Reconstitution Calculator

Background: APP

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).

References

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

Long Name

Amyloid Precursor Protein

Entrez Gene IDs

351 (Human); 11820 (Mouse)

Alternate Names

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

Related Research Areas

Citations for Recombinant Human APP/Protease Nexin II Protein, CF

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

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Anti-amyloid precursor protein immunoglobulins inhibit amyloid-beta production by steric hindrance.
Authors: Thomas RS, Liddell JE, Kidd EJ
FEBS J., 2010-12-01;278(1):167-78.
Species: Human
Sample Types: Recombinant Protein
Applications: Enzyme Assay
BACE1 and BACE2 enzymatic activities in Alzheimer's disease.
Authors: Ahmed RR, Holler CJ, Webb RL
J. Neurochem., 2009-12-04;112(4):1045-53.
Applications: Enzyme Assay Substrate