PURETIME
®
tyrosine kinase assays


Key Features Key Benefits
- Kinase and substrate additions
only
- One step
- No particles or
antibodies
- Cost effective
- Simple fluorescence intensity or fluorescence
lifetime
- Use existing reader or upgrade to robust lifetime plate reading
- Instant measurement of kinase
activity
- Kinase kinetics easily measured
Kinase assay principle

Fluorescence of PURETIME®17
is enhanced upon phosphorylation by the kinase enzyme, producing more intense emission and a longer
fluorescence lifetime. The small fluorophor does not adversely affect the action
of the kinase protein either binding ATP or phosphorylating the substrate. Fluorescence
measurements can be made at any point during the kinase incubation, permitting
time course experiments and direct determination of kinase reaction kinetics as well as
end points.
Examples
Kinase activity assay


Lyn kinase enzyme
titration performed in 384 well plate using EPEGIYGVLF-PURETIME17
substrate (500nM)
(intensity
mode above, fluorescence lifetime mode below)
Kinase
time course assay


Lck kinase time course
performed in 384 well plate using EPEGIYGVLF-PURETIME17
substrate (500nM)
(intensity
mode above, fluorescence lifetime mode below)
IC50
measurement assay

PP2 inhibition of Lyn
kinase using EPEGIYGVLF-PURETIME17
substrate (1000nM)
(IC50
found to be 6nM and 40nM at 4 micM and 40micM ATP respectively)
Fluorescence
lifetime discriminated measurements confirmed ATP analogue PP2 to exhibit
competitive inhibition of Lyn kinase. Actual IC50
values are in good agreement with published values.
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