Efficient and unbiased extraction and quantification of soil proteins - the first step to characterization
Résumé
Given the adsorption capacity of soils and the strong tendency of proteins to adsorb to surfaces, it is thus not surprising that the extraction yield of soil proteins is often poor. Given the heterogeneity of soils and the proteins they contain, it is likely that no simple recipe can ever be found to extract all proteins from all soils so that they can be quantified and characterized. We will describe some of the approaches we have used to extract soil proteins, to quantify the protein in extracts, to assess various purification steps.
In general protein desorption is favored by alkaline pH, the presence of surfactants and the addition of other proteins to compete for soil surfaces. Alkaline pH leads to the co-extraction of polyphenols that may complex with proteins and interfere with quantification and separation by SDS-PAGE. It proves difficult to separate proteins and polyphenols by the standard technique of acid precipitation and some extraction conditions appear to lead to the formation of stable complexes. Surfactants are incompatible with the commonly used Bradford assay and have to be removed prior to analysis, with the risk of loss of protein. The addition of a known protein increases soil protein extraction yield by surface exchange. Although large protein addition is not suited to a subsequent proteomic analysis of samples, this approach can be used to extract targeted proteins, for which specific assays of concentration (e.g. immune-assays) or function (e.g. catalytic activity) exist.