Nanosyn provides in vitro kinase assays based on the microfluidic mobility shift platform of PerkinElmer (former Caliper). The method reports on the enzyme activity and takes the advantage of the shift in the electrophoretic mobility of a peptide substrate upon an enzymatic conversion.
The target enzyme is incubated with fluorescently labeled substrate and test compounds in a standardized reaction mixture in 384 well plates. Upon termination of the reaction, samples are introduced onto microfluidic chips. The samples migrate through channels in the chips and product and substrate are separated based on the difference in their charge (electrophoretic mobility shift). Enzyme activity is measured by direct comparison of the fluorescence in the product and substrate peaks.
Detection of product and substrate in real time
We have the industry’s largest number of networked microfluidics instruments. Our screening and profiling services benefit from:
- Low level of false positives and false negatives
- Ratiometric approach enables high tolerance to fluorescent compounds
- Possibility to perfom real-time kinetic measurements of enzyme activity
- Capabilities to screen up to 100,000 compounds per day
- Largest portfolio (500+) of mobility shift assays run on microfluidics instruments
- Library of 400+ peptides allowing for rapid assay development
Bibliography
- Perrin D., Frémaux C., Shutes A. (2010) Capillary Microfluidic Electrophoretic Mobility Shift Assays: Application to Enzymatic Assays in Drug Discovery. Expert Opin. Drug Discov. 5, 51–63. Google Scholar
- Blackwell L.J. et al. (2009) High-Throughput Screening of the Cyclic AMP-Dependent Protein Kinase (PKA) Using the Caliper Microfluidic Platform. In: Janzen W., Bernasconi P. (eds) High Throughput Screening. Methods in Molecular Biology (Methods and Protocols), vol 565. Humana Press. Google Scholar
- Rudolf AF, Skovgaard T, Knapp S, Jensen LJ, Berthelsen J (2014) A Comparison of Protein Kinases Inhibitor Screening Methods Using Both Enzymatic Activity and Binding Affinity Determination. PLoS ONE 9(6): e98800. Google Scholar
- Janzen, William P. (2016) High throughput screening : methods and protocols Third edition. New York : Humana Press. NLM ID: 101691197
- William P. Janzen (2014) Screening Technologies for Small Molecule Discovery: The State of the Art, In Chemistry & Biology, Volume 21, Issue 9, Pages 1162-1170, ISSN 1074-5521. Google Scholar
- Yichin Liu, Raphaele Gerber, John Wu, Trace Tsuruda, John D. McCarter, High-throughput assays for sirtuin enzymes: A microfluidic mobility shift assay and a bioluminescence assay, In Analytical Biochemistry, Volume 378, Issue 1, 2008, Pages 53-59, ISSN 0003-2697. Google Scholar