Dialysis: a characterization method of aggregation tendency

Methods Mol Biol. 2015:1258:321-30. doi: 10.1007/978-1-4939-2205-5_18.

Abstract

All researchers immersed in the world of recombinant protein production are in agreement that often the production and purification process of a protein can become a nightmare due to an unexpected behavior of the protein at different protocol stages. Once the protein is purified, scientists know that they still cannot relax. There is a decisive last step missing: performing a protein dialysis in a suitable buffer for subsequent experimental trials. Here is when we can find proteins that precipitate during dialysis by buffer-related factors (ionic strength, pH, etc.), which are intrinsic to each protein and are difficult to predict. How can we find the buffer in which a protein is more stable and with less tendency to precipitate? In this chapter we go over possible factors affecting the protein precipitation tendency during the dialysis process and describe a general dialysis protocol with tricks to reduce protein aggregation. Furthermore, we propose a fast method to detect the most appropriate buffer for the stability of a particular protein, performing microdialysis on a battery of different buffers to measure afterwards precipitation by a colorimetric method, and thus being able to choose the most suitable buffer for the dialysis of a given protein.

Publication types

  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • Animals
  • Buffers
  • Dialysis / methods
  • Humans
  • Protein Aggregates / physiology*
  • Protein Stability
  • Recombinant Proteins / chemistry*

Substances

  • Buffers
  • Protein Aggregates
  • Recombinant Proteins