Organic dyes

Staining of proteins with colloidal Coomassie brilliant blue is still very popular (Chrambach et al. 1967; Patton, 2002) as it allows almost a background-free detection of proteins, a good quantitative linearity and the compatibility with mass spectrometry. Moreover, it is an easy-to-use and low-cost staining (Westermeier, 2001; Patton, 2002). Coomassie brilliant blue staining occurs in an acid environment that improves the ionic interactions between the dye and the basic amino acids moieties of the protein, and increases secondary dye-protein interactions based on hydrogen bonding, Van der Waals attraction, and hydrophobic interactions (Wirth & Romano, 1995).

Silver staining

Due to its high sensitivity, silver staining is used to visualize trace quantities of proteins of as little as one nanogram (Patton, 2002; Wirth & Romano, 1995).

Three basic silver staining procedures exist:

• diamine or ammoniacal silver stains
• chemically developed non-diamine type
• photoreduction silver stains

The silver nitrate procedure can be modified in order to make it compatible with subsequent protease digestions by removing glutaraldehyde, which otherwise cross-links the proteins and thereby inhibits the required digestion needed for mass analysis (Lopez et al. 2000; Shevchenko et al. 1996). The disadvantages of the silver stains are:

• low specificity (Steinberg et al. 1996)
• restriction to a 10-fold linear dynamic range
• complexity of the methods

all of which reduce reproducibility. For example staining has to be finished at an arbitrary time-point in order to avoid over-development of the gels, and this often results in an unsatisfactory gel-to-gel reproducibility, with variations of up to 20% in spot intensities (Patton, 2002; Quadroni & James, 1999).

Fluorescence staining

Although the fluorescent stains, such as nile red or SYPRO Red and Orange, allow a high sensitivity , with a detection limit of approx. 5 ng of protein (Alba et al. 1996; Patton, 2002), their use is not as widespread as that of Coomassie brilliant blue staining as they are:

• more difficult to handle
• more expensive (Wirth & Romano, 1995)
• require a fluorescent scanner or a charge-coupled device (CCD) camera for detection (Patton, 2002).

Fluorescent stains like SYPRO Red and Orange however offer a better specificity than silver stains, which also detect nucleic acids, lipids, lipopolysaccharides and glycolipids (Steinberg et al. 1996). In comparison to the silver-staining methods, the fluorescent dyes possess a higher reproducibility, in particular in the quantification of low intensity protein spots in a 2D gel profile. Moreover, they provide a better sequence-coverage in subsequent peptide mass profiling (Patton, 2002).

Fluorescence - based DIGE

The difference gel electrophoresis technique (DIGE) offers currently the highest reproducibility, since up to three different protein samples – individually stained - can be run together in a single gel (Tonge et al. 2001). Prior to separation of the proteins by IEF, individual samples are labelled with different dyes, such as Bimane or CyDyes, that differ in excitation and emission spectra.

By using a scanner that can excite the fluorescent dyes and measure their emission, proteins from different samples can be quantified within the same gel. The technique abolishes problems associated with shifts of the isoelectric points or molecular masses between independent gels (Unlu et al. 1997). Fluorescence labelling, however, is less sensitive than silver staining and requires a costly fluorescence scanner or CCD camera system (Alba et al. 1996).