Whoa!  Depends on the protein and the sample matrix.  HIC is short for 'hydrophobic interaction chromatography'  (a video is attached).   In short the protein is bound to the column and eluted when this bond is broken.  DEAE operates via a totally different mechanism.  So you have to give us more information.

http://www.gelifesciences.com/webapp/wcs/stores/servlet/CategoryDisplay?categoryId=3322789&catalogId=10102&productId=&top=Y&storeId=11756&langId=-1

You must always expect dilution due to dispersion unless you inject a large amount of sample and use the column to concentrate. 

Did you check each fraction of flow-through to make sure that all your proteins are well immobolized on the column?  

Several factors may lead to your loss of proteins, like

1.loss in the flow-through 

2. unsuitable condition(like pH...) and aggregations occurs or degraded

3. too strong interaction with resin to elute

4. others...

Hi there,

As already suggested above you should track your protein of interest during the chromatography process to try to figure out where the problem is as protein can't just disappear.

Let me rewind a bit. If I am understanding it well you load ammonium sulfate high concentration to a chromatography column?

If so there is in my opinion a conceptual problem as ammonium sulfate precipitates protein and then you may have your proteins "precipitated" in the column.

If you want to concentrate before putting in column you may first precipitate with ammonium sulfate, take precipitate and solubilize in small amount of "Liquid" and use a small desalting column (PD10-like column) to "put" the buffer you need for your HIC column

I apologize if I did not get well your problem.

@Rafael Franco

It is common to use buffer with ammonium sulphate (e.g. 20%) for loading on HIC column. By that you increase the hydrophobic interactions.

@Viji Viji

How do you determine your protein-of-interest? You must compare the amount before and after, not the concentration, because as it was mentioned already, the proteins get always diluted (how big are your fractions? 10s of ml? Probably more than you loaded) and it may be in more fractions. Thus first check the total amount and see whether there was really a loss.

If it's diluted, you can concentrate on any centrifugal filter or Amicon.

That said, I must also say that from my experience, HIC is not that great column, as I usually recovered only 20-30% of my protein (with about 2-fold purification I think). Ionex columns are versatile, or if you can use something specific (like affinity chromatography).

@Rafael Franco: the purpose of high concentration of salt is to decrease the solvation of proteins (without precipitating them) and therefore hydrophobic patchs become exposed for interaction with the matrix. In applying a decreasing salt gradient adsorbed proteins are eluted according to their hydrophobic properties (the less hydrophobic are eluted first).

I am sorry but there is something that does not fit in my mind. If the protein gets lost in the column it is (in my mind) because it was a dirty sample or the buffer or the eluent(s)  is/are not appropriate. 

HIC is good for hydrophobic proteins, is your protease a hydrophobic protein; has somebody used HIC to purify such protease?

Finally, I wonder why you do not use a gradient instead of replacing one solution by another of less ion strength,

By he way, I fully agree with Bruce, more info needed such as what type of sample are you putting inside the column, a crude extract, a semi-purified preparation?

Hope these thoughts may help.

Hi,

Besides issues mentioned above, one more factor could be thought of.  The purified target is protease. Is it possible to digest itself, just like unmodified trypsin?