Solubilization of Proteins in Two-Dimensional Electrophoresis

摘要: The solubilization process for 2-D electrophoresis has to achieve several parallel goals: 1. Breaking Macromolecular Interactions in Order Yield Separate Polypeptide Chains. This includes breaking disulfide bonds and disrupting all non-covalent interactions, both between proteins non-proteinaceous compounds such as lipids or nucleic acids. 2. Preventing Any Artefactual Modification of Polypeptides the Solubilization Medium. Ideally, perfect medium should freeze extracted polypeptides their exact state prior solubilization, terms amino acid composition post-translational modifications. means that enzymes able modify must be quickly irreversibly inactivated. Such include course proteases, which are most difficult inactivate, but also phosphatases, glycosidases, etc. In parallel, protocol not expose conditions chemical modifications (e.g. deamidation Asn Gin, cleavage Asp-Pro bonds) may occur. 3. Allowing Removal Substances May Interfere with Two-Dimensional Electrophoresis. 2-D, analytes. Thus, anything cell can considered an interfering substance. Many cellular coenzymes, hormones, simple sugars) do interact interfere electrophoretic process. However, many bind and/or eliminated if amount exceeds a critical interference threshold. mainly salts, lipids, polysaccharides (including walls) acids.