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λ Protein Phosphatase (λ-PPase) |
 | | | | λ Protein Phosphatase (λ-PPase) | | | |
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Prices are in US dollars and valid only for US orders.
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- Dual Specificity: protein serine/threonine/tyrosine phosphatase
- Bacteriophage λ, recombinant (E. coli)
- Supplied with 10X Reaction Buffer
Description:
Lambda Protein Phosphatase (λ-PPase) is a Mn2+-dependent protein phosphatase with activity towards phosphorylated serine, threonine and tyrosine residues. It is the 221 amino-acid product of the ORF221 open reading frame on bacteriophage lambda (1,2).
Source:
Isolated from a strain of E. coli that carries the bacteriophage lambda ORF221 open reading frame under the control of a T7 expression system (kindly provided by Dr. D. Barford) (2).
Applications:- λ-PPase can be used to release phosphate groups from serine, threonine or tyrosine residues in proteins. Note that different proteins are dephosphorylated at different rates.
Reagents Supplied:
λ-PPase Reaction Buffer Pack (10X)
Enzyme Properties
Heat Inactivation:
65°C for 1 hour
Molecular Weight:
Theoretical: 25,000 daltons
Specific Activity:
582,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X λ-PPase Reaction Buffer
Supplemented with 2 mM MnCl2
Incubate at
30°C.
1X λ-PPase Reaction Buffer:
50 mM Tris-HCl
100 mM NaCl
2 mM Dithiothreitol
0.1 mM EGTA
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:
400,000 units/ml
Storage Conditions:
50 mM HEPES
100 mM NaCl
0.1 mM MnCl2
2 mM Dithiothreitol
0.1 mM EGTA
50% Glycerol
0.01% Brij 35
pH 7.5 @ 25°C
Storage Temperature:
-70°C
Avoid repeated freeze/thaw cycles.
Notes
General notes:- λ-PPase has been purified to > 90% homogeneity as determined by SDS-PAGE and Coomassie Blue staining.
- Avoid freeze/thaw cycles Can be stored for 1 week or less at -20°C.
Usage notes:- The following information can be used as suggested initial conditions for dephosphorylation of proteins with λ-PPase.
100 units of λ-PPase 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 Serine/threonine Phosphatase (PSP) activity of λ-PPase is assessed on MyBP phosphorylated exclusively on serine/threonine residues with cAMP-dependent Protein Kinase using the PSP Assay System (NEB #P0780S). The Protein Tyrosine Phosphatase (PTP) activity is assessed on MyBP phosphorylated exclusively on tyrosine residues with Abl Protein Tyrosine Kinase using the PTP Assay System (NEB #P0785S).
λ-PPase is active on phosphorylated histidine residues (2).
0ptimal incubation times and enzyme concentrations must be determined empirically for each 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 λ-PPase (100 units) are found when the reaction buffer and MnCl2 are supplemented with:
10 mM sodium orthovanadate (3): 80%
10 mM sodium orthovanadate,
50 mM sodium fluoride: 90%
50 mM Na2 EDTA: 95%
1% Triton X-100: no
0.4% Nonidet P-40: no
0.025% Tween 20: no
0.5 M NaCl: 5%
ATP Mix (10 mM MgCl2, 0.1 mM ATP): no
Protease Inhibitor Cocktail*: 10%
*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:
λ-PPase contains no detectable protease activity.
Protease Activity:
After incubation of 10,000 units of λ-PPase with a standard mixture of proteins for 2 hours at 30ºC, no proteolytic activity could be detected by SDS-PAGE and Coomassie Blue staining.
References
- Cohen, P.T.W. and Cohen, P. (1989) Biochem. J., 260, 931-934.
- Zhuo, S. et al. (1993) J. Biol. Chem., 268, 17754-17761.
- Gordon, J.A. (1991) Methods Enzymol., 201, 477-482.
Reagents Sold Separately
λ-PPase Reaction Buffer Pack
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