In-Cell NMR in Mammalian Cells: Part 1 View Full Text


Ontology type: schema:Chapter     


Chapter Info

DATE

2012

AUTHORS

Beata Bekei , Honor May Rose , Michaela Herzig , Alexander Dose , Dirk Schwarzer , Philipp Selenko

ABSTRACT

Many mammalian IDPs exert important biological functions in key cellular processes and often in highly specialized subsets of cells. For these reasons, tools to characterize the structural and functional characteristics of IDPs inside mammalian cells are of particular interest. Moving from bacterial and amphibian in-cell NMR experiments to mammalian systems offers the unique opportunity to advance our knowledge about general IDP properties in native cellular environments. This is never more relevant than for IDPs that exhibit pathological structural rearrangements under certain cellular conditions, as is the case for human α-synuclein in dopaminergic neurons of the substantia nigra in the course of Parkinson's disease, for example. To efficiently deliver isotope-labeled IDPs into mammalian cells is one of the first challenges when preparing a mammalian in-cell NMR sample. The method presented here provides a detailed protocol for the transduction of isotope-labeled α-synuclein, as a model IDP, into cultured human HeLa cells. Cellular IDP delivery is afforded by action of a cell-penetrating peptide (CPP) tag. In the protocol outlined below, the CPP tag is "linked" to the IDP cargo moiety via an oxidative, disulfide-coupling reaction. More... »

PAGES

43-54

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/978-1-61779-927-3_4

DOI

http://dx.doi.org/10.1007/978-1-61779-927-3_4

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1029846037

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/22760311


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