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p42 MAP Kinase (Erk2) |
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- Protein serine/threonine kinase
- Mouse, recombinant (E. coli)
Description:
p42 MAP Kinase (Mitogen-Activated Protein Kinase, MAPK), also known as Erk2 (Extracellular signal-regulated kinase 2) is one of two isoforms of MAP kinase family. It is a serine/threonine protein kinase, participating in mammalian signal transduction pathways that control intracellular responses to hormones and major developmental changes. Full activation of p42 MAP Kinase requires phosphorylation at residues T183 and Y185 catalyzed by the upstream protein kinase MEK2 or MAP Kinase Kinase (MAPKK) (1-4).
Recognition Determinants: The minimal recognition motif for phosphorylation by MAPK is S/TP. Pro is also common at the -2 position in the optimal primary motif PXS/TP. The substrate specificity of MAPK overlaps with other proline-directed protein kinases present within the cell. This suggests that the recognition of protein substrates may require structural determinants in addition to primary sequence requirements (5).
Source:
Isolated from a strain of E. coli that carries a clone expressing murine MAP Kinase (1) under the control of a T7 expression system. Fully active MAP Kinase is produced by co-expression with a constitutively active form of its activator, MEK2 (6).
Reagents Supplied:
p42 MAP Kinase Reaction Buffer (10X)
Enzyme Properties
Protein Kinase Substrate Recognition
Heat Inactivation:
65°C for 20 minutes
Molecular Weight:
Theoretical: 42 kDa
Specific Activity:
10,000,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X p42 MAP Kinase Reaction Buffer
Supplemented with 200 μM ATP and 300 μCi/μmol gamma-labeled ATP
Incubate at
30°C.
1X p42 MAP Kinase Reaction Buffer:
50 mM Tris-HCl
10 mM MgCl2
2 mM DTT
1 mM EGTA
0.01 % Brij 35
pH 7.5 @ 25°C
Unit Definition:
One unit is defined as the amount of p42 MAP Kinase required to catalyze the transfer of 1 pmol of phosphate to myelin basic protein (50 µM) in 1 minute at 30°C in a total reaction volume of 30 µl.
Concentration:
100,000 units/ml
Storage Conditions:
50 mM HEPES
100 mM NaCl
1 mM DTT
0.1 mM Na2EDTA
50% Glycerol
0.01% Brij 35
pH 7.5 @ 25°C
Storage Temperature:
-70°C
Avoid repeated freeze/thaw cycles.
Notes
General notes:- p42 MAP Kinase 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.
- If the source of protein to be phosphorylated is a crude extract of cells or tissue, it is very important to include the appropriate protease and protein phosphatase inhibitors in the lysis buffer and to use shorter incubation time for phosphorylation.
- Reaction Conditions: 1X p42 MAP Kinase Reaction Buffer, supplemented with 200 µM ATP and gamma-labeled ATP to a final specific activity of 100-500 µCi/µmol.
Usage notes:- Optimal incubation times and enzyme concentrations must be determined empirically for each particular substrate.
- If possible, the ATP concentration should be at or near saturation (5 -10-fold over Km). Apparent Km values of ATP for most protein kinases are below 100 μM.
However, if the objective is to measure enzyme activity using gamma-labeled ATP, it is best to use 100-200 μM ATP in order to have higher specific activity of gamma-labeled ATP (100-500 cpm/pmol). Also, an excess of substrate should be used, and the level of phosphorylation should not exceed 10% for determination of the initial rate.
To phosphorylate a protein or peptide substrate to completion, the ATP concentration should be about 5-fold over the limited substrate concentration. Higher enzyme concentration and prolonged incubation times should be employed.
Recommended reference:
Protein Phosphorylation: A Practical Approach (1993) ed. Hardie, D.G. IRL Press.
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:
p42 MAP Kinase (Erk2) contains no detectable protease, phosphatase or MEK Kinase activities.
Contaminating Phosphatases:
After incubation of 500 units of with 50 mM p-nitrophenyl phosphate for 2 hours at 30ºC, no
phosphatase activity could be detected by spectrophotometric analysis.
MEK Kinase Assay:
After incubation of 500 units of MAP Kinase (Erk2) using unphosphorylated MAP Kinase as a substrate for 30 minutes at 30°C, no MEK Kinase activity could be detected by the phosphocellulose paper binding method.
Protease Activity:
After incubation of 500 units of MAP Kinase (Erk2) with a standard mixture of proteins for 2 hours at 30°C, no proteolytic activity could be detected by SDS-PAGE analysis.
References
- Boulton, T.G. (1991) Cell, 65, 663-675.
- Rossomando, A.J. et al. (1991) J. Biol. Chem., 266, 20270-20275.
- Payne, D.M. et al. (1991) EMBO J., 10, 885-892.
- Wu, J. et al. (1993) Mol. Cell Biol., 13, 4539-4548.
- Davis, R.J. (1993) J. Biol. Chem., 268, 14553-14556.
- Prowse, C.N. et al. (2001) J. Biol. Chem., 276, 40817-40823.
Reagents Sold Separately
p42 MAP Kinase Reaction Buffer
Companion Products
Adenosine 5´-Triphosphate (ATP)
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 5,673,244
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