Abstract
We describe a novel array on array strategy intended to enhance the throughput of enzymatic activity screening using microarrays. This strategy consists of spotting a first array with large droplets of enzymes with varying concentrations and subsequently spotting a second array with small droplets of fluorogenic substrate on top of the enzyme array. By varying the array on array spotting patterns of different classes of enzyme (e.g., proteases, phosphatases, kinases) and their corresponding fluorogenic substrates, we have the unprecedented ability for testing enzymes and mixed samples in a multiplexed fashion within a single microarray slide. This new approach enables rapid enzyme characterization building upon a one enzyme on one slide droplet-based screening concept previously established.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Foong YM, Fu J, Yao SQ, Uttamchandani M (2012) Current advances in peptide and small molecule microarray technologies. Curr Opin Chem Biol 16:234–242
Merkel JS, Michaud GA, Salcius M, Schweitzer B, Predki PF (2005) Functional protein microarrays: just how functional are they? Curr Opin Biotechnol 16:447–452
Tomizaki KY, Usui K, Mihara H (2005) Protein-detecting microarrays: current accomplishments and requirements. Chembiochem 6:782–799
Uttamchandani M, Walsh DP, Yao SQ, Chang YT (2005) Small molecule microarrays: recent advances and applications. Curr Opin Chem Biol 9:4–13
Evans MJ, Cravatt BF (2006) Mechanism-based profiling of enzyme families. Chem Rev 106:3279–3301
Uttamchandani M, Huang X, Chen GY, Yao SQ (2005) Nanodroplet profiling of enzymatic activities in a microarray. Bioorg Med Chem Lett 15:2135–2139
Babiak P, Reymond JL (2005) A high-throughput, low-volume enzyme assay on solid support. Anal Chem 77:373–377
Salisbury CM et al (2002) Peptide microarrays for the determination of protease substrate specificity. J Am Chem Soc 124:14868–14870
Gosalia DN, Salisbury CM, Ellman JA, Diamond SL (2005) High throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays. Mol Cell Proteomics 4:626–636
Gosalia DN, Diamond SL (2003) Printing chemical libraries on microarrays for fluid phase nanoliter reactions. Proc Natl Acad Sci U S A 100:8721–8726
Wang J, Uttamchandani M, Sun LP Yao SQ (2006) Activity-based high-throughput profiling of metalloprotease inhibitors using small molecule microarrays. Chem Commun (Camb) 717–719
Acknowledgments
This work was financially supported by DSO National Laboratories, Singapore.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Sieow, B.FL., Uttamchandani, M. (2017). Array-on-Array Strategy For Activity-Based Enzyme Profiling. In: Uttamchandani, M., Yao, S. (eds) Small Molecule Microarrays. Methods in Molecular Biology, vol 1518. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6584-7_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6584-7_9
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6582-3
Online ISBN: 978-1-4939-6584-7
eBook Packages: Springer Protocols