A simple, sensitive and selective quantum-dot-based western blot method for the simultaneous detection of multiple targets from cell lysates.
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.
Gilroy
Kathryn L
KL
Integrative and Systems Biology, Faculty of Biomedical and Life Sciences, University of Glasgow, University Avenue, Glasgow G12 8QQ, UK.
Cumming
Sarah A
SA
Pitt
Andrew R
AR
D015153:Blotting, Western / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D008163:Luminescent Measurements; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D045663:Quantum Dots; D013205:Staphylococcal Protein A / Q000378:metabolism*
D005456:Fluorescent Dyes; C029152:G-substrate; D007074:Immunoglobulin G; D009419:Nerve Tissue Proteins; D011506:Proteins; D013205:Staphylococcal Protein A