Miniaturized, Microarray-Based Assays for Chemical Proteomic Studies of Protein Function
Systematic analysis of protein and enzyme function typically requires scale-up of protein expression and purification prior to assay development; this can often be limiting. Miniaturization of assays provides an alternative approach, but simple, generic methods are in short supply. Here we show how custom microarrays can be adapted to this purpose. We discuss the different routes to array fabrication and describe in detail one facile approach in which the purification and immobilization procedures are combined into a single step, significantly simplifying the array fabrication process. We illustrate this approach by reference to the creation of arrays of human protein kinases and of human cytochrome P450s. We discuss methods for both ligand-binding and turnover-based assays, as well as data analysis on such arrays.
Key wordsProtein microarray Biotinylation Proteomics Functional analysis Surface capture Protein kinase Cytochrome P450 Biotin carboxyl carrier protein Inhibitor specificity
The authors thank Nashied Peton, Sarah Joyce, Colin Wheeler, Jens Koopman, Nick Workman, Steve Parham, and Mike McAndrew for their help in generating the data detailed herein. We thank Procognia Ltd (UK) for provision of human kinase arrays and also thank the Centre for Proteomic & Genomic Research, Cape Town, for access to equipment. JMB thanks the National Research Foundation (NRF; South Africa) for a Research Chair; NBK thanks the NRF for a PhD studentship. The research was supported by grants from the NRF, Procognia Ltd and Genetix PLC (UK).
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