Obtaining protein profiles of an organism is the basis for assessing several aspects of biological processes. The protein content and protein composition in haemolymph, whole body or specific tissue extracts can provide valuable information on developmental stage, reproductive potential, aging processes, health status and correlated processes. Furthermore, quantitative analysis of protein content could be the starting point for standardizing or normalizing measures on other physiological, biochemical, or morphological parameters.
The first step in any protein analysis is usually the assessment of total protein content in a given sample, so as to guide further studies, especially comparative ones. As such, accurate measurement of protein concentration is critical for any further calculations such as, representation of specific proteins in a sample and, even more so, when determining enzyme activity. Errors in the calculation of protein concentration will tend to amplify overall errors in any such further estimates.
We selected a series of classical protocols currently used for an accurate measure of protein content of samples with different natures. The simplest method for quantifying protein content is spectrophotometry at 280 nm. However, this approach is not very reliable or sensitive compared to the two principle approaches that are detailed. The first approach is the Bradford Assay, which is based on the differential binding of a staining compound (Coomassie) through ionic interactions between sulfonic acid groups and positive amine groups on proteins (Bradford, 1976). The second is the bicinchoninic acid (BCA) method, which gained importance as a means for quantification of detergent extracted protein samples (Smith et al., 1985). The Coomassie method is cheaper and very well suited for quantifying haemolymph proteins, but it is sensitive to higher detergent concentrations, as typically used for extracting proteins from tissue. In this case the BCA method is preferable. Other frequently used methods, such as that using Biuret-Folin-Ciocalteu reagents (Lowry et al., 1951) are equally sensitive as the Bradford or BCA methods, but are more laborious, and it is only for the latter reason that we do not describe the Lowry method here.
In contrast to the determination of total protein content, the analysis of protein composition can be done by a plethora of methods and their respective variants. For this reason we decided to focus on just a few which can easily be established in any laboratory with basic equipment for analytical biochemistry and using low cost reagents. These are an electrophoretic separation of proteins according to their molecular mass (actually the Stoke radius of denatured proteins), based on the original method by Laemmli (1970), and two immunological methods (Western blot analysis and rocket immunoelectrophoresis) for the detection of specific proteins in complex mixtures. Each of these methods have been frequently utilized in research on honey bees. Western blot analysis has now become a gold-standard method for identifying specific proteins, but when emphasis is on more precise quantification, rocket immunoelectrophoresis is more precise.
Notwithstanding, the methods outlined here are ones that can fairly easily be implemented in any laboratory, as they do not require sophisticated equipment. Obviously, more advanced methods are available, starting from two-dimensional electrophoresis (2DE) to ever more sophisticated and high throughput proteomics analyses. In 2DE, proteins are usually first separated by isoelectric focusing and then by SDS-PAGE in the second dimension. Such gels have much higher resolution than one-dimensional gels, and spots detected in such gels can be retrieved for amino acid sequencing by Matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF) analysis followed by comparison of amino acid sequences to proteome databases, e.g. MASCOT. 2DE methods have, for instance, been applied to study the honey bee haemolymph proteome (Chan and Foster, 2008; Boegaerts et al., 2009), and MALDI-TOF proteomics analyses have also been applied to a variety of questions in honey bee biology (Santos et al., 2005; Collins et al., 2006; Li et al., 2008).