The measurement of protein stability is essential for elucidating protein function in vivo. For example, therapeutic proteins require optimal formulation to improve shelf life. The high-throughput stability measurement presented here, rapidly tests many combinations of excipients for their effect on protein stability. The unfolding procedure is measured on a BMG LABTECH microplate reader equipped 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.
High-throughput measurement of protein stability using a BMG LABTECH microplater reader
Jean P. Aucamp, Ana M. Cosme, Gary J. Lye, Paul A. Dalby Dept of Biochemical Engineering, University College London 06/2005