Recombinant Cynomolgus Fibroblast Activation Protein alpha Summary
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||Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.|
|Shipping||The product is shipped with polar packs. 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 Tris, 1 M NaCl, 1 mg/mL BSA, pH 7.5
- Recombinant Cynomolgus Monkey Fibroblast Activation Protein alpha /FAP (cynoFAP) (Catalog # 10278-SE)
- Substrate: Z-Gly-Pro-AMC (Bachem, Catalog # I-1145), 10 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rcynoFAP to 0.2 µg/mL in Assay Buffer.
- Dilute Substrate to 100 µM in Assay Buffer.
- Load in plate 50 µL of 0.2 µg/mL rcynoFAP, and start the reaction by adding 50 µL of 100 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 100 µM Substrate.
- Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode of 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 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A9891).
- rcynoFAP: 0.01 µg
- Substrate: 50 µM
Recombinant Cynomolgus Fibroblast Activation Protein (Catalog # 10278-SE) is measured by its ability to convert the substrate benzyloxycarbonyl-Gly-Pro-7-amido-4-methylcoumarin (Z-GP-AMC) to Z-Gly-Pro and 7-amino-4-methlycoumarin (AMC).
Background: Fibroblast Activation Protein alpha/FAP
FAP (also known as seprase) is a 95 kDa Type II transmembrane serine protease that forms a homodimer and is structurally related to the dipeptidyl peptidase IV (DPPIV/CD26) family with a short cytoplasmic tail, a single transmembrane domain, and an extracellular domain that contains the active site (1-3). Within the extracellular domain, cynomolgous FAP shares 99.6% and 89.8% amino acid (aa) sequence identity with human and mouse FAP, respectively. It exhibits dipeptidyl peptidase activity with substrate specificity similar to DPPIV, which is specific for N-terminal Xaa-Pro sequences (4, 5). FAP is also an endopeptidase that can degrade Gelatin, Collagens I and IV, Fibronectin, and Laminin (1, 4, 5) as well as several peptide hormones (e.g. Neuropeptide Y, Brain Natriuretic Peptide, Substance P, Peptide YY, and Incretins) (6). FAP is also known to interact with several surface molecules to play roles in cell signaling, cell invasion and migration (3). Although not detectible in normal tissues, FAP is elevated in activated stromal fibroblasts, tumor-associated macrophages, activated hepatic stellate cells and myofibroblasts during pathological conditions that include tissue remodeling such as most types of cancer, wound healing, arthritis, atherosclerosis, and fibrosis (1, 3, 4, 7-9). Targeting FAP with vaccines, antibodies, or pharmacologics impairs tumor progression in several cancer models making it a promising immunotherapy target (9-12).
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