 |
|
 |
|
|
| Home >
Products >
Protein Tools >
Protein Phosphatases/Substrates/Inhibitors >
LAR Protein Tyrosine Phosphatase (LAR) |
 | | | | LAR Protein Tyrosine Phosphatase (LAR) | | | |
 |
|
Prices are in US dollars and valid only for US orders.
|
- Protein tyrosine phosphatase
- Human, recombinant (E. coli)
- Supplied with 10X Reaction Buffer
Description:
LAR Protein Tyrosine Phosphatase (LAR) is a phospho tyrosine-specific protein phosphatase. It consists of the 350 amino-acid soluble catalytic domain (LAR-D1) of the human transmembrane Leukocyte Antigen Related (LAR) protein tyrosine phosphatase (1,2).
Source:
Isolated from a strain of E. coli that carries the LAR-D1 fragment of the LAR protein tyrosine phosphatase cloned under the control of a T7 expression system (kindly provided by Dr. H. Saito) (1,2).
Applications:- LAR Phosphatase 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:
LAR Reaction Buffer Pack (10X)
Enzyme Properties
Heat Inactivation:
65°C for 1 hour
Molecular Weight:
Theoretical: 40,000 daltons
Specific Activity:
10,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X LAR Reaction Buffer
Incubate at
30°C.
1X LAR Reaction Buffer:
25 mM Tris-HCl
50 mM NaCl
5 mM Dithiothreitol
2 mM Na2EDTA
0.01 % Brij 35
pH 7.0 @ 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:
5,000 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:- LAR has been purified to > 95% homogeneity as determined by SDS-PAGE and Coomassie Blue staining.
- Avoid freeze/thaw cycles. Can be stored for 2 weeks or less at -20°C.
Usage notes:- The following information can be used as suggested initial conditions for dephosphorylation of proteins with LAR.
1 unit of LAR removes ~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 (PTP) activity of LAR is assessed on MyBP phosphorylated exclusively on tyrosine residues with Abl Protein Tyrosine Kinase using the PTP Assay System (NEB #P0785S).
Note that optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate.
If the source of phosphorylated protein is a crude extract of cells or tissue, it is very important to include the appropriate protease inhibitors in the lysis buffer and to use shorter incubation time for dephosphorylation.
The following levels of inhibition of LAR (1 unit) are found when the reaction buffer is supplemented with:
1 mM sodium orthovanadate (3): 100%
50 mM sodium fluoride: no
1% Triton X-100: no
0.4% Nonidet P-40: no
0.025% Tween 20: no
0.5M NaCl: no
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:
LAR contains no detectable protease activity. Tests for serine/threonine phosphatase activity showed no detectable activity.
Protease Activity:
After incubation of 100 units of LAR Protein Tyrosine Phosphatase (LAR) with a standard mixture of proteins
for 2 hours at 30ºC, no proteolytic activity could be detected by SDS-PAGE.
Serine-Threonine Phosphatase Assay:
After incubation of 100 units of LAR with phosphorylated myelin basic protein (10 µM with respect to phosphate) for 1 hour 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 #P0780S).
References
- Cho, H., Ramer, S.E., Itoh, M., Winkler, D.G., Kitas, E., Bannwarth, W., Burn, P., Saito, H. and Walsh, C.T. (1991) Biochemistry, 30, 6210-6216.
- Tsai, A.Y.M., Itoh, M., Streuli, M., Thai, T. and Saito, H. (1991) J. Biol. Chem., 266, 10534-10543.
- Gordon, J.A. (1991) Methods Enzymol., 201, 477-482.
Reagents Sold Separately
LAR Reaction Buffer Pack
|
|
|
 |
|
|
