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Abl Protein Tyrosine Kinase (Abl) |
 | | | | Abl Protein Tyrosine Kinase (Abl) | | | |
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Prices are in US dollars and valid only for US orders.
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- Protein tyrosine kinase
- Abelson Murine Leukemia Virus, recombinant (E. coli)
Description:
Abl Protein Tyrosine Kinase (AbI) is a truncated form of the v-AbI Protein Tyrosine Kinase, a partner in the Gag-Abl fusion protein of the Abelson murine leukemia virus. Abl contains 407 amino acids (residues 237-643 of the p120-gag-abl polyprotein), which include the kinase catalytic domain, SH2 domain on the N-terminus and the I237M mutation. This truncated form of v-Abl is identical to the normal c-Abl (1-3). Abl is autophosphorylated on tyrosine(s)(2).
Recognition Determinants:
The recognition motif for phosphorylation by Abl is I/V/LYXXP/F. Abl, like many cytosolic protein tyrosine kinases, preferentially phosphorylates sites recognized by its own SH2 domain, selects substrates with large hydrophobic amino acids at the +3 position and β-branched amino acids at the -1 position (4).
Source:
Isolated from a strain of E. coli that carries the truncated AbI Protein Kinase encoded by the Abelson murine leukemia virus under the control of a T7 expression system (kindly provided by Dr. S. Goff).
Reagents Supplied:
Abl Reaction Buffer (10X)
NEBuffer for Protein Kinases (PK)
Enzyme Properties
Protein Kinase Substrate Recognition
Heat Inactivation:
65°C for 20 minutes
Molecular Weight:
Theoretical: 45 kDa
Specific Activity:
13,000,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer for Protein Kinases (PK)
Supplemented with 200 μM ATP and 300 μCi/μmol gamma-labeled ATP
Incubate at
30°C.
1X NEBuffer for Protein Kinases (PK):
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 Abl required to catalyze the transfer of 1 pmol of phosphate to the Abl Peptide Substrate, EAIYAAPFAKKK (100 µM) (NEB #P6051) in 1 minute at 30°C in a total reaction volume of 25 µl (4).
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:- 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 AbI 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.
- ABI Protein Kinase phosphorylates the ABI Peptide Substrate (NEB #P6051) with a Km of 4 μM(4).
- The recombinant Abl is autophosphorylated at tyrosine residues when expressed in E. coli. The autophosphorylation was confirmed by Western blot analysis using the phospho-tyrosine specific antibody.
- 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:
Abl contains no detectable protease or phosphatase activities.
Protease Activity:
After incubation of 500 units of Abl Protein Tyrosine Kinase (Abl) with a standard mixture of proteins
for 2 hours at 30ºC, no proteolytic activity could be detected by SDS-PAGE.
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.
References
- Reddy, P.E. et al. (1983) Proc. Natl. Acad. Sci. USA, 80, 3623-3627.
- Foulkes, J.G. et al. (1985) J. Biol. Chem., 260, 8070-8077.
- Oppi, C. et al. (1987) Proc. Natl. Acad. Sci. USA, 84, 8200-8204.
- Songyang, Z. et al. (1995) Nature, 373, 536-539.
Reagents Sold Separately
Abl Reaction Buffer
Companion Products
Abl Peptide Substrate
Adenosine 5´-Triphosphate (ATP)
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