Recombinant Human DEP-1/CD148 (aa 997-1337) Protein, CF
Recombinant Human DEP-1/CD148 (aa 997-1337) Protein, CF Summary
Product Specifications
Arg997-Ala1337, with an N-terminal Met & 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
1934-DP
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol and Betamercaptoethanol. |
| Shipping | The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Assay Buffer: 10 mM HEPES, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mg/mL BSA, 1 mM dithiothreitol (DTT), pH 7.5
- Recombinant Human DEP‑1/CD148 aa 997‑1337 (rhDEP-1) (Catalog # 1934-DP)
- Substrate: Asp-Ala-Asp-Glu-Tyr(PO3)-Leu-Ile-Pro-Gln-Gln-Gly (Catalog # ES006)
- Malachite Green Phosphate Detection Kit (Catalog # DY996)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhDEP-1 to 0.00625 µg/mL in Assay Buffer.
- Load 40 µL of diluted rhDEP-1 to a plate. Include 40 µL of Assay Buffer as a Substrate Blank.
- Dilute Substrate to 1 mM and add 10 µL to all wells. The Substrate concentration is now 200 µM per well.
Cover the plate with parafilm or a plate sealer and incubate at 30 °C for 30 minutes. - Prepare a standard curve from the 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of Assay Buffer for a 10 mM stock. Continue by adding 10 µL of the 10 mM Phosphate stock to 990 µL of Assay Buffer for a 100 µM stock (this is the first dilution to use as a standard).
Perform six additional one-half serial dilutions of the 100 µM Phosphate standard stock. The standard curve has a range of 0.078 to 5 nmol per well. - After the 30 °C incubation, transfer 50 µL of the standards to the plate. Include a blank for the standard curve (50 µL Assay Buffer).
- Add 10 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
- Add 10 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- Calculate specific activity:
|
Specific Activity (μmol/min/mg) = |
Phosphate released* (nmol) x (0.001 μmol/nmol) |
| Incubation time (min) x amount of enzyme (mg) |
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Substrate.
Per Well:- rhDEP-1: 0.00000025 mg
- Substrate: 143 µM
Reconstitution Calculator
Background: DEP-1/CD148
Density Enhanced Protein Tyrosine Phosphatase (DEP-1), also known as CD148, HPTP-eta, and PTP receptor type J (PTPRJ), is an enzyme that removes phosphate groups covalently attached to tyrosine residues in proteins. A large (220 kilodalton) glycoprotein found at the cell surface, DEP-1 levels are increased with high cell density (1). DEP-1 phosphatase activity is enhanced by basement membrane proteins (2), suggesting it is involved in regulating cell adhesion and contact interactions. High levels of expression dampen PDGF (3), VEGF (4), and T-cell receptor (5) responses. DEP-1 is widely expressed in tissues, particularly ones forming epithelioid monolayers (6). In the immune system, DEP-1 is found on all cell lineages and is highest on granulocytes (7). Dep-1 is the mutated gene in the Susceptibility to Colon Cancer locus Scc1, which is altered in many human colorectal adenomas (8). Gene knockout mice lacking DEP-1 die at midgestation due to failures in cardiovascular development (9). DEP-1 dephosphorylates a variety of proteins, including the HGF (10), PDGF (11), and VEGF (4) receptors, and beta‑catenin (12). The recombinant protein is the intracellular region of DEP-1 containing the catalytic domain.
- Ostman, A. et al. (1994) Proc. Natl. Acad. Sci. USA 91:9680.
- Sorby, M. et al. (2001) Oncogene 20:5219.
- Jandt, E. et al. (2003) Oncogene 22:4175.
- Lampugnani, M.G. et al. (2003) J. Cell Biol. 161:793.
- Baker, J.E. et al. (2001) Mol. Cell. Biol. 21:2393.
- Borges, L.G. et al. (1996) Circ. Res. 79:570.
- de la Fuente-Garcia, M.A. et al. (1998) Blood 91:2800.
- Ruivenkamp, C.A. et al. (2002) Nat. Genet. 31:295.
- Takahashi, T. et al. (2003) Mol. Cell. Biol. 23:1817.
- Palka, H.L. et al. (2003) J. Biol. Chem. 278:5728.
- Kovalenko, M. et al. (2000) J. Biol. Chem. 275:16219.
- Holsinger, L.J. et al. (2002) Oncogene 21:7067.
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