Purification: chromatographic system

Exploit differences in protein properties to isolate a single type of protein from a complex mixture

 
Catherine Birck Catherine Birck IGBMC-CERBM email hidden
Anastassis Perrakis Anastassis Perrakis The Netherlands Cancer Institute email hidden
Ross Robinson Ross Robinson University of Oxford email hidden

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Purification: chromatographic system Details

Proteins of interest are usually purified from endogenous and recombinant sources using different cellular organisms or cell-free systems. Protein purification consists of a series of separation steps intended to isolate a single type of protein from a complex mixture. Separation steps may exploit differences in protein size, physicochemical properties, binding affinity and biological activity. High-performance purification is performed on preparative or analytical scale columns using chromatographic systems. Usually the proteins are detected as they are eluted from the column by their absorbance signal. Preparative purifications aim to produce a relatively large quantity of purified proteins (> 1 mg) for subsequent structural and functional studies. Analytical purifications produce a relatively small amount of proteins (0.1 - 1 mg) for a variety of research purposes and are commonly used to set up a purification protocol. Micro-purification is a technique adapted to the isolation of endogenous complexes produced in very low amount (< 0.1 mg) and difficult to express macromolecular complexes. To evaluate expression levels in multiple small test cultures and to screen for soluble or mutagenized protein constructs, a partial purification using a single affinity step may be performed on robotic systems.

User Guide

The main steps in protein purification are:

  • the cellular extraction of the macromolecules of interest and their recovery in solution by centrifugation,
  • the use of one or more affinity, gel filtration, ion-exchange and hydrophobic interaction chromatographic methods,
  • the monitoring of the purification process by running denaturing gel electrophoresis,
  • the evaluation of the purification yield and the delivery of the pure protein sample.

Standard purification protocols are available for recombinant tagged proteins which may include the removal of the tag by specific proteases. A standard purification protocol should take 2-3 days. For endogenous complexes as well as in case of protein instability, different trials may be necessary to develop an optimized purification protocol. Depending on the complexity, the set up of the optimised protocol should take several weeks.