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•Application Note: High-Throughput Measurement Of Protein Stability Using A FLUOstar OPTIMA Microplate Reader

The measurement of protein stability is essential for elucidating protein function in vivo, engineering the improved stability of therapeutic proteins or biocatalytic enzymes, and formulating proteins for therapeutic delivery. For example, therapeutic proteins require optimal formulation to improve shelf life and a highthroughput stability measurement would enable many combinations of excipients to be rapidly tested for their effect on protein stability.

High-throughput measurements of protein stability are often obtained indirectly by monitoring protein aggregation or residual activity after incubation at elevated temperatures. Both screens rely on irreversible inactivation of the protein upon unfolding, and also the assumption that positive variants result from resistance to denaturation under the test conditions. Although these indirect screens have been applied successfully, they may not easily distinguish differences in stability for proteins that spontaneously refold when returned to the activity-assay conditions.

The unfolding transition of proteins can be observed by measuring their tryptophan fluorescence upon perturbation with a chemical denaturant or a temperature shift. The average fluorescence reflects the change in local environment around the tryptophan residues and provides a direct means of assessing protein unfolding.

Here we describe a high-throughput unfolding procedure using BMG LABTECH's FLUOstar OPTIMA microplate reader with titrating syringe pump. Protein unfolding transitions were monitored by tryptophan fluorescence at 340 nm, and assessed using bovine and equine cytochrome c (cyt c), as well as bovine serum albumin (BSA) stabilized with various amounts of palmitic acid. Unfolding curves generated by the serial addition of denaturant into single wells, allowed high-throughput stability screens capable of identifying protein variants with unfolding midpoint differences of 0.15 M denaturant concentration or larger. Such a method would be suitable for screening large numbers of proteins or formulation conditions to rank the order of protein stability.

Click Here To Download:
•Application Note: High-Throughput Measurement Of Protein Stability Using A FLUOstar OPTIMA Microplate Reader

SOURCE: BMG LABTECH, GmbH