Abstract
Current chemiluminescent Western blotting technology requires sequential stripping and reprobing of the blot in order to detect more than one protein of interest. The multiple colors and long stokes shifts of quantum dot nanocrystals enable the simultaneous two‐color detection of two proteins of interest with minimal spectral overlap using a single UV or blue light excitation source on camera based imagers. WesternDot™ technology utilizes the brightest second generation quantum dots, Qdot® 625 and Qdot® 800, conjugated to streptavidin and goat anti‐mouse and goat anti‐rabbit secondary antibodies. Here we compare Qdot® 625 and Qdot® 800 conjugates to various chemiluminescent reagents. The results show that the Qdot® conjugate sensitivities and selectivities are comparable to chemiluminscent reagents, but also the work flow is faster and easier, as there is no requirement for messy chemical treatments or multiple film exposures after secondary antibody incubation. The Qdot® signal is also stable and blots can be reimaged over several weeks after processing. Our data also demonstrates that Qdot® conjugates can be effectively combined with chemiluminescent and colorometric reagents allowing for simultaneous multiplexed detection of two and three target proteins on the same blot.
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