>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Measured by its ability to dephosphorylate a tyrosine residue in a peptide containing the EGFR Y992 phosphorylation site (Catalog # ES006). The specific activity is >40 µmol/min/mg, as measured under the described conditions.
E. coli-derived human SHP-1 protein Ala205-Lys595, with an N-terminal Met and 6-His tag (C-terminal variants predicted)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
Prepare a Phosphate Standard curve by adding 10 µL of the 1 M Phosphate Standard to 990 µL of Assay Buffer for 10 mM stock. Continue by adding 10 µL of the 10 mM phosphate stock to 990 µL of Assay Buffer for 100 µM stock (this is the first dilution to use as a standard).
Perform six (6) one-half serial dilutions of the 100 µM Phosphate stock to complete the curve. The standard curve has a range of 0.078-5 nmol per well.
Dilute rhSHP-1 to 0.02 µg/mL in Assay Buffer.
Dilute Substrate to 400 µM in Assay Buffer.
Load the plate by adding 50 µL of the Phosphate Standard curve, 50 μLof Assay Buffer (Curve Blank), 25 µL of 0.02 μg/mL rhSHP‑1, and 25 μL of Assay Buffer (Substrate Blank).
Start the reaction by adding 25 µL of diluted Substrate to wells containing rhSHP-1 and to the Substrate Blank only.
Cover plate with plate sealer and incubate at 37 °C for 30 minutes.
After incubation, add 30 µL of Malachite Green reagent A to all wells used. Mix briefly.
Add 100 μL of deionized water to all wells. Mix briefly.
Add 30 µL of Malachite Green reagent B to all wells. Mix and incubate at room temperature for 20 minutes.
Read plate at 620 nm in endpoint mode.
Calculate specific activity:
Specific Activity (μmol/min/mg) =
Phosphate released* (nmol) x (.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 Blank.
rhSHP-1: 0.0000005 mg
Substrate: 200 μM
Src-Homology 2 domain Phosphatase-1 (SHP-1), also known as Protein Tyrosine Phosphatase 1C (PTP1C), PTPN6, and Hematopoetic Cell Phosphatase (HCPH), is an enzyme that selectively dephosphorylates tyrosine residues in proteins. Spontaneous point mutations in the SHP-1 gene in mice produce the “motheaten” and "motheaten viable” phenotypes that are severely autoimmune and immunodeficient (1). The enzyme is highly expressed in leukocyte cell types (2). SHP-1 has a regulatory region containing two Src homology 2 (SH2) domains that are critical for its binding to ITIM domains in inhibitory immunoreceptors (3). Deletion of the SH2 domains, as in this product, causes a marked increase in phosphatase activity (4). SHP-1 will dephosphorylate a wide variety of proteins, including the EGF receptor (5). A phosphopeptide containing the EGFR (Y992) sequence (Catalog # ES006) can be used to measure the activity of SHP-1 by detecting the release of phosphate (Catalog # DY996).
Tsui, H.W. et al. (1993) Nature Genet. 4:124.
Matthews, R.J. et al. (1992) Mol. Cell. Biol. 12:2396.
Burshtyn, D.N. et al. (1997) J. Biol. Chem. 272:13066.
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