Optimized Microscale Protein Aggregation Suppression Assay: A Method for Evaluating the Holdase Activity of Chaperones.

Many molecular chaperones act as holdases by binding hydrophobic regions of substrates to prevent aggregation. Therefore, measuring holdase activity is an amenable method to determine chaperone activity. The holdase function is reliably and easily achieved by monitoring the suppression of heat-induced aggregation of well-characterized model protein substrates. However, the standard assay format requires large amounts of protein and hence is not applicable to all proteins. Using DnaK from Escherichia coli and heat-induced aggregation of malate dehydrogenase, we describe a protocol for absorbance and fluorescence-based miniaturized versions of the standard aggregation suppression assay that are affordable and have wide application for low abundance holdases. The assay can be used for both fundamental characterization of holdase function in proteins and screening of inhibitors of holdase activity.