Samples for chromatographic purification should be clarified and free from particulate matter. Ensure that you use the right sample preparation steps.These are our 3 top tips for getting the best purity of your protein for structural studies. How do I obtain high enough purity for structural biology applications? Results could easily be misinterpreted, as the results will only show an average of the properties or miss relevant parts of the structure and only show high-level structural elements (α-helix or β-sheets). To summarize, if the level of purity of the protein of interest is not high enough (95% to 99%), the technique used for the structural study might not be possible to implement requiring time to troubleshoot and understand the cause of the issue. Impure proteins make it difficult to determine which peak/signal from the NMR is part of the target protein signal. NMR is less sensitive to impurities as each bond in the molecule contributes to the finally measured data set.In cryo-EM, poorly purified protein makes processing of EM images more difficult and the resolution of determined protein structures is lower.
Poor protein purity may also lead to the failure of protein crystals to form at all, which of course means that vital structural information will not be obtained. If the protein sample is not pure, only poorly diffracting crystals will be obtained, resulting in a low-resolution protein structure. A 3-D structure derived from a protein crystal is the average structure of the protein molecules. Millions of protein molecules are packed in a repeating array in protein crystals and weak, non-covalent forces hold the molecules together. Impurity can be caused by size or charge heterogeneity such as the co-existence of monomeric and multimeric forms of the same protein, or charge variants of a protein.
We also give you a few tips on choosing the right chromatography resin to get the highest protein purity for your chromatography protocol. This article shows why “good enough” protein purity may not be enough when you plan for structural biology study because you will need purity levels > 95%. The goal of protein purification by chromatography is of course to obtain a pure protein! But in protein research, what you do next with your pure protein-for example, determining the structure of a protein by X-ray crystallography-determines the level of protein purity that you will need.