I am thinking about using the hydrophobic column but I cannot confirm whether it would be useful or not?
Hydrophobic column tends to be more useful for proteins that have significant hydrophobic surface regions at physiologic pH. This, however, can be expanded to include extended acidic surface regions, if employing acidic conditions, where such regions will be protonated and thus uncharged (though polar). Care must be taken to ensure that the protein of interest will not be denatured under such acidic conditions.
It is better to first try to separate (at least partially) a hydrophilic protein, under physiologic conditions, using anion or cation exchange chromatography with a salt gradient. One needs to try a series of small columns to see which will be most effective for the interaction with the protein of interest. Once the right resin is identified then the process can be scaled up for preparative purposes.
Thanks Danilo Boskovic for your suggestion .Actually i am working on a thermostable protein and it is purified upto 80% by heat treatment at 80c then i purified this protein by passing through anion exchange column and then again reload this of another anion exchange column . the purified fractions always having the Ecoli proteins in both column so i reload protein then in hydrophobic column but my protein usually get bind with this column and not elute out.the problem is that the purified protein conc from anion exchange column is very less mostly protein is not purified can you help me in establishing some system on anion columns i have tried the different ph on these column but the results are always same . thankyou.
1. It is always a challenge to resolve tightly binding proteins from each other. One has to find a way of altering the binding affinity. Polar interactions can generally be modified by rising salt gradients and pH modifications. Hydrophobic interactions can be affected by reverse salt gradients and gradual introduction of detergents. The latter run the risk of denaturing the desired protein.
2. It is generally a good idea to switch column properties between steps. Have you considered following the first anion exchange column with a cation exchange column. You may be able to resolve some of the unwanted proteins.
3. Are you trying to purify a protein that was cloned into E.coli? If so , have you considered introducing a His tag to it, so that it would be easier to separate?
4. It would be helpful to know something about the protein you are working with. What is the original physiologically relevant source? What other proteins are known to bind it? Do we know the sequence? What similarities/homologies does it have?
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