Dilution of protein gel stain results in retention of staining capacity.

the invention of 2-D poly-acrylamide gel electrophoresis (2-D PAGE) (1,2) marked the beginning of what has now become a classic and dominant proteomics technology. Over the years, many refinements in the underlying technology have been made (reviewed in Reference 3), but some of the most important improvements and advancements have focused on increasing the sensitivity and visualization of the separated proteins. The techniques range from detection with scattered light following acetic acid fixation to highly sensitive silver staining techniques. In fact, there are many different silver staining techniques (4) that use more or less time-consuming protocols (5). As high-throughput and labor costs have become more and more important , the use of fluorescence dyes, following the simple " dump-and-wait-to-stain " protocol, became popular. One of the more popular commercially available staining reagents is SYPRO ® Ruby protein gel stain (Molecular Probes, Eugene, OR, USA) (6,7). SYPRO Ruby is nearly as sensitive as silver staining (8), provides the advantage of a more linear correlation between protein amount and staining intensity, yields a dramatic improvement in ease of use over traditional silver stains, and, importantly, does not interfere with mass spectrometry-based protein identification methods (9). As an example, our high-sensitivity silver staining protocol requires at least 6 h with at least 14 changes of reagents, in comparison to the SYPRO Ruby protocol , which requires 2 changes of reagents with 30 min fixation and staining overnight. However, as 2-D PAGE has become a routine practice in most laboratories now, and many investigators are employing either large-format type sys-amount of staining chemicals, and hence the cost required for any given experiment, has increased dramatically. Large-format gels can require up to 1500 mL staining solution/3 gels. The SYPRO Ruby protein gel stain-recommended staining protocol is to use the 1× stain as a ready-made solution. To evaluate whether we could reduce the cost associated with the SYPRO staining procedure without sacrificing staining sensitivity, we explored a series of stepwise dilutions of the reagent to determine the optimal concentration. Dilution of SYPRO Ruby was performed in a qualitative (2-D gels) and a quantitative way (1-D gels) using a series of dilutions, with the 1× stock as the starting concentration and diluting to a 1:20 concentration (in Milli-Q ®-quality water; Millipore, Bedford, MA, USA) of original purchased solution. Staining was performed overnight in the dark following a 30-min fixation in EtOH/acetic acid (50%/10% in Milli-Q water). Previous …