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

Abstract

Semiconductor quantum dots (Qdots) have been utilised as probes in fluorescence 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 implemenation 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 widely available), which limits the scope of applications and complicates replication. Here, the specificity of a commercially available, Qdot-conjugated secondary antibody (Qdot-Ab) was tested against several primary IgG antibodies. The 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 was found bound to only some of the protein targets in the cytosol and could not reach the nucleus, even after extensive cell permeabilisation.