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FRENCH INFRASTRUCTURE FOR INTEGRATED STRUCTURAL BIOLOGY

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INSTRUCT CENTER - FRANCE 1 - IGBMC

PURIFICATION

For purification and subsequent research purposes, the protein has to be brought into solution by breaking the tissue or cells containing it. Several methods of cell lysis are commonly used to achieve this : mechanical disruption, sonication using high frequency sound waves, liquid homogenization and freeze/thaw cycles.

The choice of the lysis method depends on the type of cells (bacteria, yeast, mammalian cells, etc), the volume of cell suspension and the fragility of the proteins to be recovered. The composition of the lysis buffer is also crucial to maintain the proteins of interest in the soluble fraction after cell breakage.

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.

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.

Devices: Akta Avent, Akta Express System, Akta purifier system