Here, we describe the single molecule force spectroscopy (SMFS)-based experimental protocol we have recently used to single out different classes of conformations in a chimeric multimodular protein containing an intrinsically disordered (human Alpha Synuclein) domain. Details are provided regarding cloning, expression and purification of the chimeric polyprotein constructs, optimal surface preparation, SMFS data collection and filtering. Although the specificity of the issue and the ensemble of nonstandard techniques needed to perform the described procedures render this a rather unorthodox protocol, it is relatively straightforward to adapt it to the study of other protein domains.

Single-molecule force spectroscopy of chimeric polyprotein constructs containing intrinsically disordered domains

TESSARI, ISABELLA;BUBACCO, LUIGI;
2012

Abstract

Here, we describe the single molecule force spectroscopy (SMFS)-based experimental protocol we have recently used to single out different classes of conformations in a chimeric multimodular protein containing an intrinsically disordered (human Alpha Synuclein) domain. Details are provided regarding cloning, expression and purification of the chimeric polyprotein constructs, optimal surface preparation, SMFS data collection and filtering. Although the specificity of the issue and the ensemble of nonstandard techniques needed to perform the described procedures render this a rather unorthodox protocol, it is relatively straightforward to adapt it to the study of other protein domains.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/2528891
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