Purifying Challenging Proteins

Abstract

This handbook is intended for students and experienced researchers with an interest in the isolation of integral membrane proteins, multiprotein complexes, or in refolding proteins from inclusion bodies. The aim is to present tools, strategies, and solutions available to meet the purification challenges associated with these three classes of proteins. For a background on techniques for protein purification, in general and handling recombinant proteins, we recommend the Recombinant Protein Purification Handbook and other handbooks in this series (see Related literature on page 100). After a general introduction to each area, high-level consensus workflows are presented to summarize current best practices in each area. Rather than providing a number of detailed protocols that have been optimized for individual proteins, this handbook provides general advice or generic protocols in a step-by-step format. The generic protocols are intended as starting points for development of separation protocols. Details will typically have to be changed to tailor the protocols for individual proteins. Furthermore, the required variations to the generic protocols cannot be predicted and unless appropriate changes are made, the protocols will only work poorly, if at allthis is one of the major challenges for the researcher involved with these groups of proteins. To address this issue, the generic protocols are presented with critical parameters identified, together with ranges of values to test for the parameters. The handbook also provides guidance, hints, and tips when using protocols other than those described here.