1.3. One-dimensional SDS gel electrophoresis of proteins
Electrophoresis is used for investigating complex mixtures of proteins by separating these according to their mobility in an electric field. It can be used to analyse subunit composition of certain proteins, to verify homogeneity of protein samples, and to purify proteins for use in further applications.
Polyacrylamide gels are the most commonly used matrices in electrophoretic separations being less costly than agar or agarose gels and providing a very broad range of options for defining matrix pore size (the sieving properties of the gel) through selecting appropriate proportions of polyacrylamide/bisacrylamide and water in the gel mix. In polyacrylamide electrophoresis (PAGE), proteins are separated according to charge and molecular mass/molecule structure. The use of sodium dodecyl sulphate (SDS) as detergent for eliminating differences in charge, the reduction of disulfide bonds by treatment with a reducing reagent, such as ß-mercaptoethanol or dithiothreitol (DTT) and heat to denature protein structure, were innovations by Laemmli (1970). This created the widely used SDS-PAGE protocols for separating proteins according to their Stoke’s radius, commonly referred to as molecular mass (not molecular weight, as weight is dependent on gravity).
Unlabelled proteins separated by PAGE are typically detected by staining either with Coomassie Brilliant Blue or with silver salts. Coomassie Brilliant Blue binds nonspecifically to proteins but not the gel, thereby allowing visualization of the proteins as discrete blue bands within a translucent gel matrix. Observe that Coomassie Brilliant Blue G-250 is used in the Bradford assay, but it is Coomassie Brilliant Blue R-250 which is used for staining gels. These are different reagents, so be careful to use the correct one for each application.
Silver staining, although more laborious, is significantly more sensitive, but it may present problems when quantification of protein bands in gel documentation systems is the aim. The coloration of silver-stained bands is not uniform and bands of high protein content may in fact invert colour intensity and appear transparent.
Although there are a plethora of variations of the original SDS-PAGE protocol (Laemmli, 1970) adapting the method to specific problems, we describe a protocol commonly used to assess the haemolymph composition (Fig. 1) of honey bee larvae and adults (Pinto et al., 2000; Barchuk et al., 2002; Guidugli et al., 2005; Bitondi et al., 2006). A major variation in this protocol compared to the original Laemmli protocol is that there is no SDS in the gel, but only in the buffers. This avoids the precipitation of SDS in the gel matrix when running thin gels at low temperatures, conditions used to improve separation. After protein staining with Coomassie Brilliant Blue and scanning the gels on a gel documentation system, it is further possible to quantify specific proteins by Image J software (http://rsbweb.nih.gov/ij/index.html) or by commercial software implemented in gel documentation systems. Gels stained with silver salts are not appropriate for such quantitative analyses as silver staining does not follow linear characteristics.
Fig. 1. Haemolymph protein patterns of Apis mellifera workers separated by SDS-PAGE (7.5 %), lane 1: 4-day-old worker; lane 2: 6-day-old worker; lane 3: molecular mass marker. Gel stained with Coomassie Brilliant Blue. Modified from Bitondi and Simões (1996). Copyright Journal of Apicultural Research.