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

• Published in: Analytical and bioanalytical chemistry 2010-09, Vol.398 (1), p.547-554
• Main Authors: Gilroy, Kathryn L, Cumming, Sarah A, Pitt, Andrew R
• Format: Article
• Language: English
• Subjects: Analytical Chemistry; Antibodies; Binding; Biochemistry; Biological; Blotting, Western - methods; Cellular; Cellular proteins; Characterization and Evaluation of Materials; Chemical and thermal methods; Chemiluminescence; Chemistry; Chemistry and Materials Science; Exact sciences and technology; Fluid flow; Fluorescent Dyes; Food Science; Humans; Immunoglobulin G; Immunoglobulin G - immunology; Laboratory Medicine; Luminescent Measurements; Mathematical analysis; Methods; Monitoring/Environmental Analysis; Multiplexing; Nerve Tissue Proteins - metabolism; Original Paper; Protein A/G; Protein binding; Proteins - analysis; Proteins - metabolism; Quantum Dots; Qunatum dots; Staphylococcal Protein A - metabolism; Western blotting
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-010-3908-0

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. [graphic removed]