The solubilization process for 2D electrophoresis has to achieve four parallel goals:1. Breaking macromolecular interactions in order to yield separate polypeptide chains.This includes denaturing the proteins to break noncovalent interactions, breaking disulfide bonds, and disrupting noncovalent interactions between proteins and non-proteinaceous compounds such as lipids or nucleic acids.2. Preventing any artefactual modification of the polypeptides in the solubilization medium.Ideally, the perfect solubilization medium should freeze all the extracted polypeptides in their exact state prior to solubilization, both in terms of amino acid composition and in terms of post-translational modifications. This means that all the enzymes able to modify the proteins must be quickly and irreversibly inactivated. Such enzymes include of course proteases, which are the most difficult to inactivate, but also phosphatases, glycosidases etc. In parallel, the solubilization protocol should not expose the polypeptides to conditions in which chemical modifications (e.g. deamidation of Asn and Gln, cleavage of Asp-Pro bonds) may occur.3. Allowing the easy removal of substances that may interfere with 2D electrophoresis.In 2D, proteins are the analytes. Thus, anything in the cell but proteins can be considered as an interfering substance. Some cellular compounds (e.g. coenzymes, hormones) are so dilute they go unnoticed. Other compounds (e.g. simple non-reducing sugars) do not interact with proteins or do not interfere with the electrophoretic process. However, many compounds bind to proteins and/or interfere with 2D, and must be eliminated prior to electrophoresis if their amount exceeds a critical interference threshold. Such compounds mainly include salts, lipids, polysaccharides (including cell walls) and nucleic acids.
Keeping proteins in solution during the 2D electrophoresis process.Although solubilization stricto sensu stops at the point where the sample is loaded onto the first dimension gel, its scope can be extended to the 2D process per se, as proteins must be kept soluble till the end of the second dimension. Generally speaking, the second dimension is a SDS gel, and very few problems are encountered once the proteins have entered the SDS PAGE gel. The one main problem is overloading of the major proteins when micropreparative 2D is carried out, and nothing but scaling-up the SDS gel (its thickness and its other dimensions) can counteract overloading a SDS gel. However, severe problems can be encountered in the IEF step. They arise from the fact that IEF must be carried out in low ionic strength conditions and with no manipulation of the polypeptide charge. IEF conditions give problems at three stages: a.During the initial solubilization of the sample, important interactions between proteins of widely different pI and/or between proteins and interfering compounds (e.g. nucleic acids) may happen. This yields poor solubilization of some components.
b.During the entry of the sample in the focusing gel, there is a stacking e...