Purification: chromatographic system

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

 
Catherine Birck Catherine Birck email hidden
Anastassis Perrakis Anastassis Perrakis email hidden
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Purification: chromatographic system Details

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.

Technical Specifications

Workstations with automated liquid handling modules are used for high throughput parallel protein purification. Different chromatographic systems are available for large, analytical and micro-scale purification of proteins and macromolecular complexes using a variety of prepacked columns and chromatography media. The flow rates are flexible in the range 0.001 ml/min to 10ml/min depending on the pump and column specificities. Samples are loaded manually on the column using a syringe or automatically using a loading pump. Proteins are detected as they are eluted from the column by their UV absorption at multiple wavelengths in the range 190-700 nm. Samples are collected in microplates or in a variety of test tube sizes. Fixed volume fractionation or automatic peak fractionation is possible.