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Issue 6, 2012
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The importance of adding EDTA for the nanopore analysis of proteins

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Abstract

Nanopore analysis is a promising technique for studying the conformation of proteins and protein/protein interactions. Two proteins (bacterial thioredoxin and maltose binding protein) were subjected to nanopore analysis with α-hemolysin. Two types of events were observed; bumping events with a blockade current less than −40 pA and intercalation events with blockade currents between −40 pA and −100 pA. In potassium phosphate buffer, pH 7.8, both proteins gave intercalation events but the frequency of these events was significantly reduced in TRIS or HEPES buffers especially in the presence of 0.01 mM divalent metal ions. The frequency of events was restored by the addition of EDTA. For maltose binding protein, the frequency of intercalation events was also decreased in the presence of maltose but not lactose to which it does not bind. It is proposed that the events with large blockade currents represent transient intercalation of a loop or end of the protein into the pore and that divalent metal ions inhibit this process. The results demonstrate that the choice of buffer and the effects of metal ion contamination are important considerations in nanopore analysis.

Graphical abstract: The importance of adding EDTA for the nanopore analysis of proteins

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Publication details

The article was received on 05 Mar 2012, accepted on 17 Apr 2012, published on 30 Apr 2012 and first published online on 30 Apr 2012


Article type: Paper
DOI: 10.1039/C2MT20050C
Citation: Metallomics, 2012,4, 539-544
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    The importance of adding EDTA for the nanopore analysis of proteins

    B. Krasniqi and J. S. Lee, Metallomics, 2012, 4, 539
    DOI: 10.1039/C2MT20050C

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