Fluorescence Gel Imaging: Take a Walk on the Bright Side Application Note · UVP GelStudio

Introduction

Several DNA and protein detection reagents are available to researchers that vary in sensitivity, safety, and suitability for downstream applications. Chief among DNA binding dyes is ethidium bromide with detection limits as low as 0.5 ng1. Ethidium bromide, or homidium as referred to in the veterinary sciences, was used to effectively treat trypanosomiasis in cattle as early as the 1950s, for which the mechanism of action was unknown until recently2. As any molecular biologist would have expected, the dye interferes with trypanosome DNA replication2, underscoring the mutagenic safety concerns with this and other DNA-binding dye technologies (See our Safety is a Part of Science application note). While ethidium bromide has fallen out of favor in the treatment of cattle for the aforementioned risks, ethidium bromide-agarose gel electrophoresis still remains the most common fluorescence-based DNA detection method since its conception3. However, several alternative fluorescence-based DNA dyes are available to investigators (See Figure 1 and Table 1).

For protein, colorimetric dyes remain the most common. Coomassie Brilliant Blue (CBB) is arguably the most popular post-electrophoresis protein dye. Several iterations of the original protocol exist with reported detection limits as low as 3 ng4. Alternatively, silver salt staining can increase the detection limits of protein as low as 0.1 ng. Typically, silver staining achieves 100x-1000x more sensitivity than CBB in routine protocols5. Still, both methods either require large volumes of toxic reagents or laborious and time-consuming staining protocols. Fortunately, several bright fluorescence-based protein dyes are available to investigators that have vastly streamlined in-gel protein detection (Table 1).