628280-41-1Relevant academic research and scientific papers
An Integrated Chemical Cytometry Method: Shining a Light on Akt Activity in Single Cells
Mainz, Emilie R.,Wang, Qunzhao,Lawrence, David S.,Allbritton, Nancy L.
supporting information, p. 13095 - 13098 (2016/10/30)
Tools to evaluate oncogenic kinase activity in small clinical samples have the power to guide precision medicine in oncology. Existing platforms have demonstrated impressive insights into the activity of protein kinases, but these technologies are unsuitable for the study of kinase behavior in large numbers of primary human cells. To address these limitations, we developed an integrated analysis system that utilizes a light-programmable, cell-permeable reporter deliverable simultaneously to many cells. The reporter's ability to act as a substrate for Akt, a key oncogenic kinase, was masked by a 2-4,5-dimethoxy 2-nitrobenzyl (DMNB) moiety. Upon exposure to ultraviolet light and release of the masking moiety, the substrate sequence enabled programmable reaction times within the cell cytoplasm. When coupled to automated single-cell capillary electrophoresis, statistically significant numbers of primary human cells were readily evaluated for Akt activity.
Long-wavelength fluorescent reporters for monitoring protein kinase activity
Oien, Nathan P.,Nguyen, Luong T.,Jernigan, Finith E.,Priestman, Melanie A.,Lawrence, David S.
supporting information, p. 3975 - 3978 (2014/05/06)
In vivo optical imaging must contend with the limitations imposed by the optical window of tissue (600-1000 nm). Although a wide array of fluorophores are available that are visualized in the red and near-IR region of the spectrum, with the exception of proteases, there are few long wavelength probes for enzymes. This situation poses a particular challenge for studying the intracellular biochemistry of erythrocytes, the high hemoglobin content of which optically obscures subcellular monitoring at wavelengths less than 600 nm. To address this, tunable fluorescent reporters for protein kinase activity were developed. The probing wavelength is preprogrammed by using readily available fluorophores, thereby enabling detection within the optical window of tissue, specifically in the far-red and near-IR region. These agents were used to monitor endogenous cAMP-dependent protein kinase activity in erythrocyte lysates and in intact erythrocytes when using a light-activatable reporter. Searching far and near: Fluorescent reporters for protein kinase activity were developed. The probing wavelength is preprogrammed by using readily available fluorophores, thereby enabling detection within the optical window of tissue, specifically in the far-red and near-IR region. These agents were used to monitor endogenous cAMP-dependent protein kinase activity 1) in erythrocyte lysates and 2) in intact erythrocytes when using a light-activatable reporter.
Fluorescent assays for protein kinases
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, (2010/08/04)
This invention provides fluorescently-labeled peptide substrates for protein kinases; methods using the substrates for identifying compounds that inhibit protein kinases, for determining if particular protein kinases are active in cells, for diagnosing di
A Light-Activated Probe of Intracellular Protein Kinase Activity
Veldhuyzen, Willem F.,Nguyen, Quan,McMaster, Gary,Lawrence, David S.
, p. 13358 - 13359 (2007/10/03)
The first example of a photoactivated probe of intracellular enzymatic activity is described. The caged derivative of a fluorescent protein kinase C peptide-based sensor was prepared by modifying the free hydroxyl group of a phosphorylatable serine moiety with a photolabile appendage that blocks phosphoryl transfer. We have demonstrated that the caged sensor allows one to (1) sample PKC activity with exquisite temporal precision, (2) control the relative amount of active sensor available for phosphorylation, and (3) examine protein kinase activity at multiple time points. Copyright
