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Author Topic: PROTEIN PURIFICATION TECHNIQUES  (Read 4940 times)
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Francis Umeoguaju
Expert in Bioscience Issues
Posts: 657

« on: April 10, 2010, 08:32:59 am »

Protein purification as with every other purification aims, at isolating a particular protein from a mixture comprising thousands of protein. The purification of protein employs the existence of various differences between proteins as a basis for their separation. Such differences include solubility, density, size, charge etc. some of the commonly used purification techniques are discussed below.


Protein filtration as with every other purification. It is used to remove  debris from crude homogenate.
It separates proteins based on differences in size. Commonly used filtering apparatus includes filter paper, Muslin cloth and
Buckner funnel filtering apparatus.


This is used to obtain a clear filtrate. It gives the clear soluble content of an homogenate. Depending on the treatment given to the homogenate, the sediment or the supernatant may be obtained for further purification.


This separates protein based on their ability to precipitate at a particular concentration of the precipitating agent.

Usually when a clear supernatant is obtained, the supernatant is believed to be rich in many soluble protein. A specific concentration of the precipitating agent is used to precipitated proteins of interest since different proteins have varying ability to precipitate at a given concentration of the precipitating agent. The solution is then centrifuged and the pellet is re-dissolved in the solvent that was initially used to extract the protein. This method can be used to concentrate the protein.

Common solvent used for precipitation includes Acetone and Ammonium sulphate. Usually after precipitation and re-dissolving, the solution is dialyzed against a suitable buffer to remove contaminating ions such as NH4+, ammonium contents may also be removed by desalting the protein extract in a G25 chromatographical column


This separates proteins based on the differential ability of the protein to bind to an adsorbent material at some specific conditions. The differential binding of proteins is the direct consequence of the presence of various charged groups on proteins. A common adsorbent material used for this purpose is the calcium hydroxyl apartite.

Using the Hydroxyl Apartite; Usually, this is done by thoroughly mixing a known volume of the extract with a given weight of the gel (hydroxyl apartite). The mixture is then centrifuged. The supernatant is decanted or discarded depending on whether the protein of interest is bound or unbound. The pellet is then washed with distilled water two to three times to remove those protein that are poorly bound to the gel.

Some proteins binds so tightly that they require a buffer with strong ionic strength before they can be eluted. The bound proteins may then be differentially washed off the gel.

The differential binding affinity of proteins is the basis of use of hydroxyl apartite as a purification agent.

Gel filtration

This separates protein based on the difference in their molecular size. The gel filtration column is usually packed with gels such as sephadex, sephrose and Biogel P etc depending on the molecular size the protein. For instance sephadex G-75 is used for fractionating proteins in the range of the relative molecular mass range of 30000 to 70000 (walker et al 1992).

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