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Wednesday, January 11, 2012

Protein Desalting and Concentration

Protein desalting is done to remove salts from a protein solution, the salts may come from the buffers or from the biological sample solutions. Protein desalting (removal of salts) is important while its coming to the analysis part. Salts can interfere with the analytical techniques. Separation techniques like electrophoresis, HPLC (high performance liquid chromatography), Mass Spectrometry etc requires protein solutions without salts so protein desalting becomes important steps before using these techniques. The presence of salts and other low molecular weight compounds can increase the ionic strength and there by decreasing the resolution of electrophoresis and also it gives false peak in the chromatograms.
The presence of proteins in blood, serum, urine and other biological samples are used for diagnostic purpose, in which elevated levels of particular protein or other compounds indicates specific disease state.concentrating protein is also very important, since most of the times proteins in the samples are in minute quantities this might go undetected during diagnosis and other analysis steps.

Methods for Protein Desalting

Desalting columns

Protein Desalting column

There are many methods for protein desalting and concentration, these includes dialysis, molecular sieve chromatography (Gel Filtration), HPLC, diafiltration, ultrafiltration, Ion Exchange, freeze drying, etc.
Gel Filtration is one of the best method for desalting protein solutions. Gel filtration matrix has beads having pores, based on the size, molecules get separated.Gel filtration Principle and method can be found from the previous posts.
Examples for gel filtration matrix includes, sephadex G-25, Biogel P-30, etc.
Diafiltration is another method used for protein desalting and protein concentration, here the solution is passed through a filter with specific pore size which retains salts and allow other components to pass through it. Complete desalting wont be achieved in single step. Membrane systems are available from Millipore, Pall etc. Even small amounts of protein solution can be desalted and buffer exchanged.
Ion Exchange is another method for desalting protein, here ion exchange resin is used as stationary phase, protein desalting can be achieved using this method.
For protein desalting and concentration certain columns are available like zeba column, centricons, which can be centrifuged and result in faster desalting and concentration.