Evaluation of quantum dot conjugated antibodies for immunofluorescent labelling of cellular targets

Abstract

Semiconductor quantum dots (Qdots) have been utilised as probes in fluorescent microscopy and provide an alternative to fluorescent dyes and fluorescent proteins, due to their brightness, photostability, and the possibility to excite different Qdots with a single wavelength. In spite of these attractive properties, their take up by biologists has been somewhat limited and only a few Qdot conjugates are commercially available for the labelling of cellular targets. Although, many protocols have been reported for the specific labelling of proteins with Qdots, the majority of these relied on Qdot-conjugated antibodies synthesised specifically by the authors and therefore not broadly available, which limits the scope of applications and complicates replication. Here, the specificity of a commercially available Qdot conjugated secondary antibody (Qdot-Ab), for different antigens, was tested. Antigens were labelled simultaneously with a fluorescent dye coupled to a secondary antibody (Dye-Ab) and the Qdot-Ab. Although, the Dye-Ab labelled all of the intended target proteins, the Qdot-Ab only bound to some of the protein targets in the cytosol and could not reach the nucleus even after extensive cell permeabilisation.