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

Kathryn L. Gilroy, Sarah A. Cumming, Andrew R Pitt

Research output: Contribution to journalArticle

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.
Original languageEnglish
Pages (from-to)547-54
Number of pages8
JournalAnalytical and Bioanalytical Chemistry
Volume398
Issue number1
Early online date27 Jun 2010
DOIs
Publication statusPublished - Sep 2010

Keywords

  • Western blotting
  • fluorescent dyes
  • humans
  • immunoglobulin G
  • luminescent measurements
  • nerve tissue proteins
  • proteins
  • quantum dots
  • staphylococcal protein A

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