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PhosTRAK ™ : A Homogeneous 3-Minute Phosphatase Assay. Michael J. Corey, Ph.D. Current Affiliation: CellExSys, Inc. How Shall We Measure Phosphatase Activity?. Detect dephosphorylated product There’s no good, fast way to do this Usually complex operationally Tends to be expensive
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PhosTRAK™: A Homogeneous3-Minute Phosphatase Assay Michael J. Corey, Ph.D. Current Affiliation: CellExSys, Inc.
How Shall We Measure Phosphatase Activity? • Detect dephosphorylated product • There’s no good, fast way to do this • Usually complex operationally • Tends to be expensive • Unnatural substrates--easy to run, difficult to interpret • Detect free phosphate • Malachite Green assay is slow, insensitive • Other methods are operationally complex • Background is a problem
PhosTRAK Philosophy • Time, space, and labor are valuable • Instrument time is valuable • Statistical significance is predictive • Natural substrates are good • Two steps are sometimes one too many
Side-by-Side Comparison of PhosTRAK and Malachite Green PhosTRAK Malachite Green Limit of Detection 3-10 pmol 40-125 pmol Assay time 3 minutes 20-90 minutes Operational characteristics Mix, read Mix, wait, add reagent, wait, read
Homogeneous PhosTRAK Assay ofl-Phosphatase: Substrate Dependence
Detection of Calcineurin Activity by PhosTRAK (Timepoint Assay) 60’ reaction, 30oC
Inhibition of Calcineurin by Autoinhibitory Peptide (Measured with PhosTRAK) 15’ preincubation, 17’ reaction
Autoactivation of Native Calcineurin Observed by Homogeneous PhosTRAK No Enzyme +Native Bovine Calcineurin
PhosTRAK Operational Characteristics • Start to read immediately after initiation (3 minutes plenty) • Today’s luminometers can read a plate in 2 seconds • Cocktail • On ice, full cocktail slowly converts dynamic background to static (Pi to ATP)--otherwise fine for two hours • Stable if enzyme cocktail is withheld and added before assay • Data reduction • Linear fit if you don’t have automatic injector • Single read fine if you do • Waste--no problem
Challenges and Drawbacks • Phosphate contamination of G3P is responsible for most of the background--should be an easy separation • Must avoid other sources of phosphate • Molecules that inhibit the coupled enzymes can yield false hits • Inhibitor and validation data are lacking
Possibilities • Use in HTS for phosphatase inhibitors--targeted libraries appear especially suitable • (Hypothetical) Measurement of T-cell activation, using either CD45 or calcineurin • Phosphate monitoring in various systems
Summary • PhosTRAK is a 3-minute homogeneous phosphatase assay suitable for use in HTS with many protein phosphatases • Based on phosphate detection; uses natural substrates • No waste issue • Requires luminometer
Acknowledgments • Eva Corey • POWERPOINT EXPERT • (Also Assistant Professor in Urology at UW) • Bob Kinders (Alidex, Inc.) and Bob Vessella (UW) for facilities and advice
Contact Information Michael J. Corey CellExSys, Inc. 1100 Olive Way, Suite 100 Seattle, WA 98101 206-521-4846 Email: coreym@targen.com