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Study Notes: Protein Purification

Protein bands of interest may be easily recovered from gels and purified. In fact, running a mixed protein sample on a gel is a purification step since a well separated and discrete protein band has been purified away from all the other material in the sample. There may well be closely migrating or co-migrating species in the band but most of the other material has been removed and the protein band is assumed to be pure.

A very simple method for extracting the protein band is to stain the gel and excise the protein band from the gel using a scalpel blade. The gel fragment containing the protein band is then crushed and soaked in a suitable buffer and electroeluted.

A schematic diagram showing the steps involved in protein purification.
Schematic diagram showing the steps involved in protein purification

Fixing and staining the protein band reduces the recovery of the protein from the gel significantly. There are a couple of ways around this, as follows.

  • Use radioactively labelled proteins and expose a piece of X-ray film to the wet gel (messy but achievable with plastic wrapping). The developed X-ray film is then used as a template to cut the band of interest from the wet gel.
  • Use a guide strip technique. A duplicate lane is run, excised from the gel, fixed and stained as usual then used as a guide to locate the band of interest in the unstained part of the gel.

A schematic diagram showing the guide strip technique.
The guide strip technique

If staining is unavoidable then elution of the crushed gel fragments into a buffer containing SDS or urea will help to solubilise the protein aggregates.

Gel apparatus is also available with a special stacking gel comb that produces only one long and deep sample well. In this circumstance there is much more starting sample material loaded, more material in the long protein band across the gel and even with relatively poor recoveries, an adequate amount of the purified protein can be obtained for further purposes.

Gel Elution
Gel elution using the crush and soak method is very effective. Forcing small gel fragments through a syringe needle is very effective in breaking up the gel. The gel particles are then suspended in a buffer containing 4 - 8M urea or 0.1% SDS. The buffer is chosen to be compatible with the required use of the protein.

Electroelution
Electroelution is used to concentrate the sample. Dialysis or the use of concentration columns may also be used.

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