Linked Life Data

17105162

Source:http://linkedlifedata.com/resource/pubmed/id/17105162

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
22
pubmed:dateCreated
2006-11-19
pubmed:abstractText
We have extended the method of single-molecule fluorescence, two-color coincidence detection (TCCD) to detect coincident events due to a low fraction of a complex against a background of chance coincident events, due to monomers. We developed two complementary methods to determine the number of chance coincident events using the experimental data and without the need for additional experiments. We show that the subtraction of the chance coincidence level is essential for accurate quantification of the relative number of complexes and their stoichiometry. By performing experiments on model samples made from fluorophore-labeled duplex DNA and free dye, a linear dependence on the fraction of duplex DNA was found, independent of the level or ratio of free dye, with quantification down to a level of 0.5% and 500 fM duplex DNA. The method was then used to measure the equilibrium dissociation constant and offrate of a 9-mer duplex DNA, demonstrating the application of this method to systems with nanomolar dissociation constants. These improvements to the method of TCCD analysis significantly extend the application of TCCD to weakly bound complexes and large multicomponent biomolecular systems.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
0003-2700
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
78
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
7707-15
pubmed:meshHeading
pubmed:year
2006
pubmed:articleTitle
Determination of the fraction and stoichiometry of femtomolar levels of biomolecular complexes in an excess of monomer using single-molecule, two-color coincidence detection.
pubmed:affiliation
Department of Chemistry, Cambridge University, Lensfield Road, Cambridge, CB2 1EW, UK.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't