C(O)[C:2]([NH2:7])([CH2:5]O)[CH2:3][OH:4].Cl.[Mg+2].[Cl-].[Cl-].P(OC[C@H]1O[C@@H](N2C3N=CN=C(N)C=3N=C2)[C@H](O)[C@@H]1O)(OP(OP(O)(O)=O)(O)=O)(=O)[OH:14].C(N(CC(O)=O)CC(O)=O)COCCOCCN(CC(O)=O)CC(O)=O.SC[C@H]([C@@H](CS)O)O.CCCCCC[CH2:84][CH2:85][CH2:86][CH2:87][CH2:88][CH2:89][O:90]CCO>>[NH2:7][C@H:2]([C:3]([OH:4])=[O:14])[CH2:5][C:86]1[CH:85]=[CH:84][C:89]([OH:90])=[CH:88][CH:87]=1
An Abl reaction mixture was prepared by mixing 1 ml of reaction buffer (50 mM Tris-HCl, 10 mM MgCl2 1 mM EGTA 2 mM dithiothreitol (pH 7.5 at 25° C.)), 10 μl of 10 mM ATP, 2 μl of 6-(9-oxo-9H-acridin-10yl) hexanoate-labelled Abl Peptide substrate (non-phosphorylated) (500 μM in DMSO). 100 μl of this mixture was placed into the wells of a black flat bottomed microtitre plate. The reaction was initiated by the addition of 10 μl of Abl kinase (New England Biolabs, Code P6050L Lot 5), (100,000 units/ml or 100 units/μl) which had been diluted in reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM EGTA, 2 mM dithiothreitol, 0.01% Brij 35 (pH 7.5 at 25° C.) to a concentration of 100 units per 10 μl.
Wird geladen…
