Recombinant Human FAM20C Protein, CF Summary
with a C-terminal 6-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||Supplied as a 0.2 μm filtered solution in Tris and NaCl.|
|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.
- Universal Kinase Activity Kit (Catalog # EA004)
- Assay Buffer: 25 mM HEPES, 150 mM NaCl, 10 mM MnCl2, 10 mM CaCl2, pH 7.0
- Recombinant Human FAM20C (rhFAM20C) (Catalog # 9265-FM)
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute 1 mM Phosphate Standard provided by the Universal Kinase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
- Complete the standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock using Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
- Dilute ATP (supplied in kit) to 0.4 mM in Assay Buffer.
- Dilute rhFAM20C to 33.34 ng/µL in Assay Buffer.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
- Load 15 µL of 33.34 ng/µL rhFAM20C into empty wells of the same plate as the curve. Include a Control containing 15 μL of Assay Buffer.
- Add 25 µL of 0.4 mM ATP into all wells, excluding the standard curve.
- Seal plate and incubate at 37 °C for 1 hour.
- Dilute Coupling Phosphatase 4 (supplied in kit) to 10 ng/µL in Assay Buffer.
- Add 10 µL of 10 ng/µL Coupling Phosphatase 4 to all wells, excluding the standard curve.
- Seal and incubate plate at room temperature for 5 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
- Add 100 µL of deionized water to all wells. Mix briefly.
- Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate sealed plate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Phosphate released* (nmol) x (1000 pmol/nmol)|
Incubation time (min) x amount of enzyme (µg)
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.
** Based upon a 65 minute incubation time. (No coupling rate constant.)
- rhFAM20C: 0.5 µg
- Coupling Phosphatase 4: 0.1 µg
- ATP: 0.2 mM
Fam20C is a protein kinase dedicated to the phosphorylation of extracellular proteins, including caseins, and is localizes within the Golgi apparatus and also is secreted (1, 2). It phosphorylates the caseins as well as several other secreted proteins such as AMELX, AMTN, ENAM, SPP1 and the small integrin-binding ligand N-linked glycoproteins SIBLINGs implicated in biomineralization (3). Consequently, mutations in Fam20C cause an osteosclerotic bone dysplasia in humans known as Raine syndrome (4) and hypophosphatemic rickets (5). The discovery of the function of Fam20C has solved a 130-year-old mystery of the identity of the kinases that phosphorylate caseins and has shed light on several human disorders of biomineralization (6). In addition to its role in biomineralization, Fam20C also plays a role in lipid homeostasis, wound healing and cell migration and adhesion (1).
- Tagliabracci, V. S. et al. (2013) cell 161:1619.
- Tagliabracci, V. S. et al. (2012) Science 336:6085.
- Cui, j. et al. (2015) Elife 4:e06120
- Fradin M, et al. (2011) Clin. Genet. 80:177.
- Wang X, et al. (2012) PLoS Genet. 8(5):e1002708.
- Tagliabracci VS, et al. (2013) Trends Biochem. Sci. 38:121.
Citations for Recombinant Human FAM20C 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 - 3
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Detection and Characterization of Phosphorylation, Glycosylation, and Fatty Acid Bound to Fetuin A in Human Blood
Authors: M Ková?ová, H Kalbacher, A Peter, HU Häring, T Didangelos, N Stefan, A Birkenfeld, E Schleicher, K Kantartzis
Journal of Clinical Medicine, 2021;10(3):.
Sample Types: Protein
Secretory Pathway Kinase FAM20C, a Marker for Glioma Invasion and Malignancy, Predicts Poor Prognosis of Glioma
Authors: S Du, S Guan, C Zhu, Q Guo, J Cao, G Guan, W Cheng, P Cheng, A Wu
Onco Targets Ther, 2020;13(0):11755-11768.
Sample Types: Whole Cells
Ras-transformation reduce FAM20C expression and osteopontin phosphorylation
Authors: GN Schytte, B Christense, I Bregenov, ES Sørensen
Biosci. Rep., 2020;40(9):.
Sample Types: Reference Standard
Applications: Western Blot
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