Modern proteomics applications are mainly based on the combination of biochemical separation techniques with mass spectrometric analysis methods.

The 1- or 2-dimensional separation of protein mixtures via gel electrophoresis is one of the most commonly used separation methods. After staining, the bands/ spots are excised and digested for subsequent identification by MS-analysis and database search. In many cases, qualitative protein identification is of interest, another issue may be the quantitative comparison of different protein patterns, e.g. when it comes to «differential display» analyses.
For the outcome of such experiments, the staining of the proteins on the electrophoresis gels is still of striking importance. Traditional protein stains like Coomassie Brillant Blue have the advantage of easy handling and good MS-compatibility, but often lack the desired sensitivity. Silver stains on the other hand are extremely sensitive, but show disadvantages due to a narrow linear range of quantification, restricted MS-compatibility, huge time consumption, complicated handling and laborious waste management.
Newer developments in the field of fluorescence dyes provide advantageous alternatives. They are sensitive, have a large linear range for quantification and are MS-compatible. Even for the simple staining of 1D-gels, fluorescence dyes offer the advantages of time-reduction and being easy to use.
In this article, we report on the application profiles of 3 recently developed fluorescence dyes for the sensitive detection of proteins on electrophoresis gels: LUCY-506, LUCY-565 and LUCY-569.
We analysed the following parameters:
n sensitivity and limit of detection
n linear dynamic range
n different staining protocols
n optimal electrophoresis conditions
n time-amount and handling
n detection and visualization
n compatibility with subsequent MS-analysis
– LUCY-506 shows highest sensitivity, with a detection limit of around 3 ng protein per band. The standard procedure is a post-electrophoretic stain with omission of the fixation step. The stain is completed after 60min, and detection is possible immediately thereafter.
– LUCY-565 is recommended if after staining, a westernblot transfer should be performed from the same gel. This is possible, because staining is done under neutral, non-fixing conditions. With traditional dyes, 2 gels and therefore the double amount of sample material have been necessary for that.
– LUCY-569 suits particularly well for protein quantification. It has an extraordinary large linear dynamic range between 10-6000 ng/band, which is larger than for most silver staining methods [1], Coomassie Brillant Blue or other fluorescence dyes.
All the 3 dyes are «stains», i.e. no covalent-binding «labels». The adsorption to the protein is done via the SDS-coating, which must not be removed by organic solvents after the electrophoresis run. Accordingly, even native gels, which were run under SDS-free conditions, may be visualized by incubating them directly after the run in SDS. Subsequent staining is done according to the standard protocol.
For the determination of MS-compatibility, protein bands were excised from LUCY-stained gels, washed according to common protocols, and digested using trypsin. The resulting peptides were mixed with HCCA matrix, loaded onto a MALDI-sample-plate and measured via MS.
Various possibilities are suitable for detection, depending on the lab equipment, e.g. illuminating the gels on a transilluminator with the corresponding wavelength (Dark-Reader™; UV-Screen) and imaging using a CCD-camera plus filter. Alternatively, a laser scanner may be used, with the respective excitation- and emission filter settings, corresponding to the spectral data of the used dye.
The 3 dyes are delivered as 5000-fold stock solutions in DMSO, which are stored cooled in the dark. ®

References:
[1] White et al., Electrophoresis 25(17), 3048-3054 (2004)