详细说明
- Purity>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
- Endotoxin Level<1.0 EU per 1 μg of the protein by the LAL method.
- ActivityMeasured by its ability to hydrolyze the 5’-phosphate groups from the substrate adenosine-5’-triphosphate (ATP). The orthophosphate product is measured by a Universal Kinase Activity Kit (Catalog # ). The specific activity is >20 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .
- SourceChinese Hamster Ovary cell line, CHO-derived Val63-Arg584 with a C-terminal 6-His tag
- Accession #
- N-terminal Sequence
AnalysisVal63 (pro-mature) and Asp93 (mature) - Predicted Molecular Mass60 kDa
- SDS-PAGE70-82 kDa, reducing conditions
9265-FM | | |
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.
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- Universal Kinase Activity Kit (Catalog # )
- 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 # )
- 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.)
Per Reaction:
- rhFAM20C: 0.5 µg
- Coupling Phosphatase 4: 0.1 µg
- ATP: 0.2 mM
- References:
- 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.
- Long Name:Family with Sequence Similarity 20, Member C
- Entrez Gene IDs:56975 (Human); 80752 (Mouse); 304334 (Rat)
- Alternate Names:dentin matrix protein 4; DKFZp547C074; DMP4; DMP-4; FAM20C; family with sequence similarity 20, member C; GEF-CK; RNS