Many key protein functions require the protein to undergo a post-translational modification (PTM), examples ranging from phosphorylations and glycosylations to histone modifications and ubiquitinations. HuProt human proteome microarrays can be used for profiling the targets of enzymes producing PTMs and have been used in studies of phosphorylation, glycosylation, sumoylation, etc.
The arrays bring the advantage of monitoring modification on a proteome-wide scale in a single experiment. They enable screening the targets of purified enzymes or whole cell extracts, where changes in modification pattern may associate with disease. Kinase assays were first performed on yeast protein arrays (a fore-runner of the HuProt arrays) to define the yeast kinome (Ptacek J. et al. Nature 2005; 438:679-84). In practice, the enzyme performing the modification is incubated on the array and the interaction is monitored by detection of the modified proteins, often using a PTM-specific antibody.
Assay setup
The on-array modification is indicated by the red triangles. For array readout by fluorescence, the result of enzyme activity needs to be converted to a fluorescent signal, e.g. the PTM introduced by the enzyme of interest can be detected by an anti-PTM antibody. Assay requirements are as diverse as the enzymes – please contact us to discuss the options and develop a strategy for your project.
See references here for specific examples of PTM detection on HuProt arrays.
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Contact us to discuss how your screening project can benefit from using HuProt v4 arrays or the set-up options for your protein.