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A simple, sensitive and selective quantum-dot-based western blot method for the simultaneous detection of multiple targets from cell lysates

Gilroy, K.L., Cumming, S.A., and Pitt, A.R. (2010) A simple, sensitive and selective quantum-dot-based western blot method for the simultaneous detection of multiple targets from cell lysates. Analytical and Bioanalytical Chemistry, 398 (1). pp. 547-554. ISSN 1618-2642 (doi:10.1007/s00216-010-3908-0)

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Abstract

Quantum dots (Qdots) are fluorescent nanoparticles that have great potential as detection agents in biological applications. Their optical properties, including photostability and narrow, symmetrical emission bands with large Stokes shifts, and the potential for multiplexing of many different colours, give them significant advantages over traditionally used fluorescent dyes. Here, we report the straightforward generation of stable, covalent quantum dot-protein A/G bioconjugates that will be able to bind to almost any IgG antibody, and therefore can be used in many applications. An additional advantage is that the requirement for a secondary antibody is removed, simplifying experimental design. To demonstrate their use, we show their application in multiplexed western blotting. The sensitivity of Qdot conjugates is found to be superior to fluorescent dyes, and comparable to, or potentially better than, enhanced chemiluminescence. We show a true biological validation using a four-colour multiplexed western blot against a complex cell lysate background, and have significantly improved previously reported non-specific binding of the Qdots to cellular proteins.

Item Type:Article
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Gilroy, Dr Kathryn and Cumming, Dr Sarah and Pitt, Dr Andrew
Authors: Gilroy, K.L., Cumming, S.A., and Pitt, A.R.
Subjects:Q Science > QH Natural history > QH345 Biochemistry
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Analytical and Bioanalytical Chemistry
ISSN:1618-2642
ISSN (Online):1618-2650
Published Online:27 June 2010

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
387191Radical Solutions for Researching the Proteome (RASOR)Andrew PittBiotechnology and Biological Sciences Research Council (BBSRC)BB/C511572/1Institute of Molecular Cell and Systems Biology