Recombinant Mouse BACE-1 Protein, CF Summary
Thr22-Thr457, with a C-terminal 10-His tag
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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.
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.
|Formulation||Lyophilized from a 0.2 μm filtered solution in Tris and NaCl.|
|Reconstitution||Reconstitute at 100 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.
- Assay Buffer: 50 mM Sodium Acetate, 100 mM NaCl, pH 4.0
- Recombinant Mouse BACE-1 (rmBACE-1) (Catalog # 2976-AS)
- Substrate: MCA-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Arg-Lys(DNP)-Arg-Arg-NH2 (Catalog # ES004)
- Heparin (Sigma, Catalog # H3393), 20 mg/mL in deionized water
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rmBACE-1 to 20 ng/µL in Assay Buffer.
- Dilute Heparin to 4 ng/μL in Assay Buffer.
- Combine equal volumes diluted Heparin and rmBACE-1.
- Incubate at 37 °C for 30 minutes.
- Dilute Substrate to 20 μM in Assay Buffer.
- Load 50 µL of the rmBACE-1/heparin mixture in a plate and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 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.
- Calculate specific activity:
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).
- rmBACE-1: 0.500 µg
- Substrate: 10 µM
- Heparin: 1 ng/μL
BACE-1 is an aspartic protease and an integral membrane protein (1, 2). It is the major beta secretase, and together with the gamma secretase, is responsible for generating the amyloid beta peptide (A beta ) from the amyloid precursor protein (APP) (3, 4). Because A beta is a major component of amyloid plaques, BACE-1 has been implicated in the onset and/or progression of Alzheimer's disease. High levels of BACE-1 activity are sufficient to elicit neurodegeneration and neurological decline in vivo, indicating that inhibiting BACE-1 may block not only A beta -dependent but also A beta -independent pathogenic mechanisms (5). In addition to APP, BACE-1 also cleaves APP-like proteins 1 and 2, the cell adhesion protein P-selectin glycoprotein ligand-1 and beta -galactoside alpha 2,6-sialyltransferase, implying that BACE-1 may have additional functions involving the ectodomain shedding of membrane proteins (6-8). The purified recombinant mouse BACE-1 corresponds to the ectodomain with the activity as described in Activity Assay Protocol.
- Vassar, R. et al. (1999) Science 286:735.
- Yan, R. et al. (1999) Nature 402:533.
- Cai, H. et al. (2001) Nature Neurosci. 4:233.
- Roberds, S.L. et al. (2001) Human Mol. Genet. 97:1317.
- Rockenstein, E. et al. (2005) J. Biol. Chem. 280:32957.
- Li, Q and T.C. Sudhof (2004) J. Biol. Chem. 279:10542.
- Lichtenthaler, S.F. et al. (2003) J. Biol. Chem. 278:48713.
- Kitazynem, S. et al. (2005) J. Biol. Chem. 280:8589.
Citations for Recombinant Mouse BACE-1 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.
Citations: Showing 1 - 2
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HtrA2 regulates beta-amyloid precursor protein (APP) metabolism through endoplasmic reticulum-associated degradation.
Authors: Huttunen HJ, Guenette SY, Peach C, Greco C, Xia W, Kim DY, Barren C, Tanzi RE, Kovacs DM
J. Biol. Chem., 2007;282(38):28285-95.
Sample Types: Whole Cells
The ACAT inhibitor CP-113,818 markedly reduces amyloid pathology in a mouse model of Alzheimer's disease.
Authors: Hutter-Paier B, Huttunen HJ, Puglielli L, Eckman CB, Kim DY, Hofmeister A, Moir RD, Domnitz SB, Frosch MP, Windisch M, Kovacs DM
Applications: Enzyme Assay
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Fluorogenic Peptide Substrates
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