Recombinant Human FAP/His (Catalog # 3715-SE) has a molecular weight (MW) of 176.3 kDa as analyzed by SEC-MALS, suggesting that this protein is a homodimer. MW may differ from predicted MW due to post-translational ...read more
Recombinant Human FAP His-tag (Catalog # 3715-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-methylcoumarin (AMC).
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-methylcoumarin (AMC). The specific activity is >1800 pmol/min/µg, as measured under the described conditions.
Source
Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Fibroblast Activation Protein alpha/FAP protein Leu26-Asp760, with an N-terminal 6-His tag
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.
Applications/Dilutions
Dilutions
Enzyme Activity
Theoretical MW
86 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
85 kDa, reducing conditions
Publications
Read Publications using 3715-SE in the following applications:
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
6 months from date of receipt, -70 °C as supplied.
3 months, -70 °C under sterile conditions after opening.
Buffer
Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Assay Procedure
Assay Buffer: 50 mM Tris, 1 M NaCl, 1 mg/mL BSA, pH 7.5
Recombinant Human Fibroblast Activation Protein alpha /FAP (rhFAP) (Catalog # 3715-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 rhFAP to 0.2 µg/mL in Assay Buffer.
Dilute Substrate to 100 µM in Assay Buffer.
Load 50 µL of 0.2 µg/mL of rhFAP into a plate, and start the reaction by adding 50 µL of 100 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate.
Read at excitation and emission wavelengths of 380 nm and 460 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 7-Amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).
Per Well:
rhFAP: 0.010 µg
Substrate: 50 µM
Notes
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human FAP Protein, CF
170 kDa melanoma membrane-bound gelatinase
DKFZp686G13158
DPPIV
EC 3.4.21.-
FAP
FAPA
Fibroblast Activation Protein alpha
fibroblast activation protein, alpha
Integral membrane serine protease
Seprase
vibronectin
Background
FAP (also known as seprase) is a 95 kDa Type II transmembrane serine protease that is structurally related to dipeptidyl peptidase IV (DPPIV/CD26) (1, 2). Within the extracellular domain (ECD), human FAP shares 90% amino acid (aa) sequence identity with mouse and rat FAP (3, 4). Alternative splicing of human FAP generates a truncated isoform that consists of the C-terminal 239 residues of the ECD. A soluble and enzymatically active form of FAP known as antiplasmin-cleaving enzyme (APCE) circulates in human plasma (4). FAP is expressed on reactive stromal fibroblasts in tumor tissue and wound healing and on synoviocytes in rheumatoid arthritis (1, 6-8). It exhibits dipeptidyl peptidase activity with substrate specificity similar to DPPIV, which is specific for N-terminal Xaa-Pro sequences (6, 9). FAP is also an endopeptidase that can degrade Gelatin, Collagens I and IV, Fibronectin, and Laminin (1, 6, 9) as well as several peptide hormones (e.g. Neuropeptide Y, Brain Natriuretic Peptide, Substance P, Peptide YY, and Incretins) (10). The enzymatic activity is dependent on FAP association with DPPIV on the cell surface (6, 9, 11, 12). The matrix-dedgrading activity of FAP contributes to tumor cell migration and invasion (11-14). In addition, FAP can enhance tumor cell growth by limiting the development of anti-tumor immunity (15).
Zi, F. et al. (2015) Mol. Med. Rep. 11:3203.
Pineiro-Sanchez, M.L. et al. (1997) J. Biol. Chem. 272:7595.
Niedermeyer, J. et al. (1997) Int. J. Cancer 71:383.
Scanlan, M.J. et al. (1994) Proc. Natl. Acad. Sci. USA 91:5657.
Park, J.E. et al. (1999) J. Biol. Chem. 274:36505.
Rettig, W.J. et al. (1988) Proc. Natl. Acad. Sci. USA 85:3110.
Bauer, S. et al. (2006) Arthritis Res. 8:R171.
Aertgeerts, K. et al. (2005) J. Biol. Chem. 280:19441.
Keane, F.M. et al. (2011) FEBS J. 278:1316.
Ghersi, G. et al. (2006) Cancer Res. 66:4652.
Ghersi, G. et al. (2002) J. Biol. Chem. 277:29231.
Cheng, J.D. et al. (2005) Mol. Cancer Ther. 4:351.
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