• 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.  

• Activity

  Measured 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 .

• Source

  Chinese Hamster Ovary cell line, CHO-derived Val63-Arg584 with a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Val63 (pro-mature) and Asp93 (mature)

• Predicted Molecular Mass

  60 kDa

• SDS-PAGE

  70-82 kDa, reducing conditions

Assay Procedure

Materials

• 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

1. 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.

2. 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.

3. Dilute ATP (supplied in kit) to 0.4 mM in Assay Buffer.

4. Dilute rhFAM20C to 33.34 ng/μL in Assay Buffer.

5. Load 50 μL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.

6. 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.

7. Add 25 μL of 0.4 mM ATP into all wells, excluding the standard curve. 

8. Seal plate and incubate at 37 °C for 1 hour.

9. Dilute Coupling Phosphatase 4 (supplied in kit) to 10 ng/μL in Assay Buffer.

10. Add 10 μL of 10 ng/μL Coupling Phosphatase 4 to all wells, excluding the standard curve.

11. Seal and incubate plate at room temperature for 5 minutes.

12. Add 30 μL of the Malachite Green Reagent A to all wells. Mix briefly.

13. Add 100 μL of deionized water to all wells. Mix briefly.

14. Add 30 μL of the Malachite Green Reagent B to all wells. Mix and incubate sealed plate for 20 minutes at room temperature.

15. Read plate at 620 nm (absorbance) in endpoint mode.

16. Calculate specific activity:

     *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

Background: FAM20C

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).

• References:

1. Tagliabracci, V. S. et al. (2013) cell 161:1619.

2. Tagliabracci, V. S. et al. (2012) Science 336:6085.

3. Cui, j. et al. (2015) Elife 4:e06120

4. Fradin M, et al. (2011) Clin. Genet. 80:177.

5. Wang X, et al. (2012) PLoS Genet. 8(5):e1002708.

6. 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