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Research
Thermal denaturation by circular dichroism is still the gold standard for determining protein stability. However, because of the lengthy procedure it’s not the ideal method for screening libraries of designed mutants. A medium throughput screen for chemical denaturation was developed as a microplate based assay monitoring tryptophan fluorescence. After gene assembly of the designed library, colonies are picked, cultured and the histidine tagged proteins are purified in a 96 well format by Ni-NTA filter plates. Mutant proteins are then denatured by guanidinium chloride on 96 well microplates. Well to well standard deviation is very good (1-4%), however plate to plate values are not, thus requiring the wild type protein to be present on every plate as a reference. Library sizes of 48 and 96 members have been screened by this procedure, providing a wealth of protein stability data. The method developed here can be used to screen the stability of any library of histidine tagged mutants, provided their primary structure features a tryptophan residue. Instant applications of this method exist in computational and combinatorial-based protein engineering and protein folding.
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