期刊
JOURNAL OF BIOMOLECULAR SCREENING
卷 14, 期 6, 页码 700-707出版社
SAGE PUBLICATIONS INC
DOI: 10.1177/1087057109335749
关键词
thermal shift assays; protein unfolding; protein stabilization; superoxide dismutase
资金
- Medicines for Malaria Venture
- NIAID [P01AI067921]
- NIH [GM64655]
- UNDP/World Bank/WHO [05-00508]
In the past decade, thermal melt/thermal shift assays have become a common tool for identifying ligands and other factors that stabilize specific proteins. Increased stability is indicated by an increase in the protein's melting temperature (T(m)). In optimizing the assays for subsequent screening of compound libraries, it is important to minimize the variability of T(m) measurements so as to maximize the assay's ability to detect potential ligands. The authors present an investigation of T(m) variability in recombinant proteins from Plasmodium parasites. Ligands of Plasmodium proteins are particularly interesting as potential starting points for drugs for malaria, and new drugs are urgently needed. A single standard buffer (100 mM HEPES [pH 7.5], 150 mM NaCl) permitted estimation of T(m) for 58 of 61 Plasmodium proteins tested. However, with several proteins, T(m) could not be measured with a consistency suitable for high-throughput screening unless alternative protein-specific buffers were employed. The authors conclude that buffer optimization to minimize variability in T(m) measurements increases the success of thermal melt screens involving proteins for which a standard buffer is suboptimal. (Journal of Biomolecular Screening 2009:700-707)
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