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TABLE OF CONTENTS
I.
BEFORE STARTING
5
II.
INTENDED USE
8
III.
PRINCIPLE OF THE ASSAY
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IV.
ASSAY DEVELOPMENT
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A. Deciding on substrate configuration
B. Deciding on assay format
C. Titration of biotinylated phosphopeptide and/or
non-phosphorylated peptide substrate
D. Evaluating optimal enzymatic assay conditions
E. Termination of the kinase reaction
F. Kinetics of the detection reaction
G. Titration of PhosphoSensor Acceptor beads
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11
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V.
SUMMARY OF ASSAY DEVELOPMENT
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VI.
DETAILED PROTOCOLS
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A. Titration of phosphorylated versus
non-phosphorylated substrate
B. All-in-one-well and transfer kinase assays
C. AlphaScreen beads quality control
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VII.
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TROUBLESHOOTING GUIDE
3