Microstructure optimization of alumina/zirconia materials resistant for abrasive wear

Abstract

Protein aggregation cause artifacts in most biophysical techniques, leads to un-certainty in the active concentration of protein in solution and loss of biological activity. Unfortunately, it is a common problem for many protein constructs when removed from their biological context (e.g. solution conditions, domain boundaries, post-translational modifications, chaperones, interaction partners), and present at high concentrations for measurements. Detection of Aggregates Aggregation can be detected or inferred in various ways e.g.: • Visual observation of particulate matter in suspension. • Detection of very large species eluting in the void-volume during size ex-clusion chromatography. • Detection of abnormally high light-scattering in absorbance measurements. • Detection of very large particles by Dynamic Light Scattering. • Inference from loss of activity • Inference from experimental artifacts, the nature of which depends on the type of measurement and the technique. Screening for Thermal Stability vs Screening against Aggregation A popular general approach to increase protein stability is to employ dye-based thermal shift assays to screen for buffer conditions or additives that increase the thermal denaturation midpoint of the protein. This is a good approach if one is primarily concerned to ensure thermal stability, i.e. that the protein essentially exclusively populates the native (folded) state at a given temperature. Screening to prevent aggregation isn't necessarily so straightforward. If the pro-tein aggregates only from the denatured (unfolded) state, then thermally sta-bilising the protein should prevent aggregation. This is because thermal stabil-isation will decrease the concentration of the denatured state – the aggregating species – at a given temperature. However, if aggregation occurs from the native state, an intermediate state, or an apo-state, thermal stabilisation may not help at all. It may in fact increase the concentration of the aggregating state(s). One can screen more specifically for conditions that prevent aggregation by Dynamic Light Scattering, or by a simple light-scattering assay in an absorbance plate reader. Scattering from particulate aggregates in suspension can give an increase in apparent absorbance in the range 300-600 nm over time.