High-throughput thermal scanning: a general, rapid dye-binding thermal shift screen for protein engineering.

ABSTRACT: The low stability of natural proteins often limits their use in therapeutic, industrial, and research applications. The scale and throughput of methods such as circular dichroism, fluorescence spectroscopy, and calorimetry severely limit the number of variants that can be examined. Here we demonstrate a high-throughput thermal scanning (HTTS) method for determining the approximate stabilities of protein variants at high throughput and low cost. The method is based on binding to a hydrophobic dye akin to ANS, which fluoresces upon binding to molten globules and thermal denaturation intermediates. No inherent properties of the protein, such as enzymatic activity or presence of an intrinsic fluorophore, are required. Very small sample sizes are analyzed using a real-time PCR machine, enabling

SUBMITTER: Lavinder JJ 

PROVIDER: S-EPMC2701314 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature