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YOP Protein Tyrosine Phosphatase (YOP PTP) |
 | | | | YOP Protein Tyrosine Phosphatase (YOP PTP) | | | |
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Prices are in US dollars and valid only for US orders.
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- Protein tyrosine phosphatase
- Yersinia enterocolitica, recombinant (E. coli)
- Supplied with 10X Reaction Buffer
Description:
YOP Protein Tyrosine Phosphatase (YOP PTP) is a phospho tyrosine-specific protein phosphatase. It is the product of the Yop51 gene of Yersinia enterocolitica, that contains the C235R mutation (Yop51*)(1,2).
Source:
Isolated from a strain of E. coli that carries the YOP protein tyrosine phosphatase coding sequences under the control of a T7 expression system (kindly provided by Dr. J.E. Dixon) (2).
Applications:- YOP PTP can be used to release phosphate groups specifically from phospho tyrosine residues in proteins. Note that different proteins are dephosphorylated at different rates.
Reagents Supplied:
NEBuffer for Protein Tyrosine Phosphatases (PTP)
YOP Reaction Buffer (10X)
Enzyme Properties
Heat Inactivation:
65°C for 1 hour
Molecular Weight:
Theoretical: 51,000 daltons
Specific Activity:
125,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer for Protein Tyrosine Phosphatases (PTP)
Incubate at
30°C.
1X NEBuffer for Protein Tyrosine Phosphatases (PTP):
50 mM Tris-HCl
100 mM NaCl
5 mM DTT
2 mM Na2EDTA
0.01 % Brij 35
pH 7.5 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme that hydrolyzes 1 nmol of p-nitrophenyl phosphate (50 mM) in 1 minute at 30°C in a total reaction volume of 50 µl.
Concentration:
200,000 units/ml
Storage Conditions:
50 mM HEPES
100 mM NaCl
5 mM Dithiothreitol
2 mM EDTA
50% Glycerol
0.01% Brij 35
pH 7.0 @ 25°C
Storage Temperature:
-70°C
Avoid repeated freeze/thaw cycles.
Notes
General notes:- YOP PTP has been purified to > 95% homogeneity as determined by SDS-PAGE and Coomassie Blue staining.
Usage notes:- Avoid repeated freeze/thaw cycles. Can be stored for 2 weeks or less at -20°C.
The following information can be used as suggested initial conditions for dephosphorylation of proteins with YOP PTP.
50 units of YOP PTP remove ~100% of phosphates in phosphorylated myelin basic protein (phospho-MyBP) in 30 minutes in a 50 µl reaction. The concentration of phospho-MyBP is 5 µM with respect to phosphate.
The Protein Tyrosine Phosphatase activity of YOP PTP is assessed on MyBP phosphorylated exclusively on tyrosine residues with Abl Protein Tyrosine Kinase using the PTP Assay System (NEB #P0785S).
Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate.
YOP PTP is inhibited by vanadate (2).
The following levels of inhibition of YOP PTP (50 units) are found when the reaction buffer is supplemented with:
1 mM sodium orthovanadate (3): 100%
50 mM sodium fluoride: no
1% Triton X-100: 5%
0.4% Nonidet P-40: no
0.025% Tween 20: no
0.5 M NaCl: 10%
Protease Inhibitor Cocktail*: no
*Pepstatin A, leupeptin and aprotinin, 10 µg/ml each, 0.5 mM PMSF, and 1 mM benzamidine
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
YOP PTP contains no detectable protease activity. Tests for serine/threonine phosphatase activity showed no detectable activity.
Protease Activity:
After incubation of 1,000 units of YOP Protein Tyrosine Phosphatase (YOP PTP) with a standard mixture of proteins
for 2 hours at 30ºC, no proteolytic activity could be detected by SDS-PAGE.
Serine-Threonine Phosphatase Activity:
After incubation of 1,000 units of YOP PTP with phosphorylated myelin basic protein (10 µM with respect to phosphate) for 30 minutes no serine-threonine phosphatase activity could be detected by release of radioactivity.
Myelin basic protein was phosphorylated exclusively on serine/threonine residues with cAMP-dependent Protein Kinase using the Protein Serine/Threonine Phosphatase Assay System (NEB #P0780).
References
- Guan, K. and Dixon, J.E. (1990) Science, 249, 553-556.
- Zhang, Z.-Y., Clemens, J.C., Schubert, H.L., Stuckey, J.E., Fischer, M.W.F., Hume, D.M., Saper, M.A. and Dixon, J.E. (1992) J. Biol. Chem., 267, 23759-23766.
- Gordon, J. A. (1991) Methods Enzymol., 201, 477-482.
Reagents Sold Separately
NEBuffer for Protein Tyrosine Phosphatases (PTP)
YOP Reaction Buffer
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