Acetone precipitation is very likely to cause loss of all activity. There are three simple options for desalting your protein after re-suspension of the ammonium sulfate pellet: (1) gel exclusion chromatography, which can be done with a hand-operated desalting cartridge, (2) repeated diliution with buffer and concentration in a centrifugal ultrafilter, or dialysis against several changes of salt-free buffer. For any method, you should clarify the re-suspended pellet by centrifugation before proceeding. Ultrafilters should be PES, not cellulose acetate or nylon, as these may strongly bind certain proteins, and may be the cause of the loss of activity. We typically do #1 for our protein preps where salt precipitation is a purification step.

As far as I know, there are two ways for desalting.. gel filtration and using membrane (either dialysis or electrodialysis). If you want a fast method, electrodialysis would work. Dialysis usually require an overnight procedure, while electrodialysis will be faster, as it aided by electricity.

Or, if you want to concentrate your protein, but don't want any salt in it, you can try to precipitate using organic solvent such as acetone..

Acetone precipitation is very likely to cause loss of all activity. There are three simple options for desalting your protein after re-suspension of the ammonium sulfate pellet: (1) gel exclusion chromatography, which can be done with a hand-operated desalting cartridge, (2) repeated diliution with buffer and concentration in a centrifugal ultrafilter, or dialysis against several changes of salt-free buffer. For any method, you should clarify the re-suspended pellet by centrifugation before proceeding. Ultrafilters should be PES, not cellulose acetate or nylon, as these may strongly bind certain proteins, and may be the cause of the loss of activity. We typically do #1 for our protein preps where salt precipitation is a purification step.

You seem to have lost your protein in the filtration step before applying it on the HiTrap column, right? I would assume that you had aggregates too big for the filter and thus lost the protein. I would suggest dialysis after precipitating the protein with salt, that usually worked fine for me.

You must first check the solubility of the protein you want to purify (at which pH (what is the pHi of the protein?), at which ionic strength (it may have been salted out) etc.

Addition of known amounts of a protein (BSA for exemple) may help to keep the protein biologically active. The protein added should be choosen to facilitate its elimination when necessary.

Addition of small amounts of DMSO may help, without denaturating the protein (1-8% in vol).

The most efficient way I used to eliminate and concentrate a protein is first desalting on a small column equilibrated with the appropriate buffer -pH, ionic strength- (ready to use or self prepared regarding the amount of proteins) and ultrafiltration on a centrifuge device

There are various materials that can compose the 0.2 micron filters. Your protein apparently sticks to the kind of filter you used. Try some other filter media to see what binds your protein. Centrifugal concentration filters and disposable microdialysis/concentration devices are alternatives to the syringe-based filters.

I taught these procedures in a proteomics workshop lecture/lab at Iowa State University, Department of Biochemistry, Biophysics and Molecular Biology for 6 years. I prefer the classical protein chemistry methodology. The class did not work with maize extracts, but soy meal extracts (and E. coli extracts), a big difference, in protein content as I remember. So, a crude extract, you mention. But you did not say how the extract was obtained, a very important point. Assume you have the extract [using urea? (fresh urea of course), .5 M sodium/potassium chloride? maybe .1 M sodium citrate?] all help to solubilize complexes with otther components. Then centrifuge to clarify (12,000 x g), dialyze in the cold with 3 buffer exchanges vs 5 mM ammonium bicarbonate, then salt out with ammonium sulfate, using solid crystals, very carefully and slowly. Let sit for at least 15-20 minutes or longer in an icebucket if you don't see a precipitate, collect by centrifugation, redisslove in minimal volume of 5 mM ammonium bicarbonate , and dialyze vs the the same, three changes over 12-24 hours in the cold. Then clarify, which means centrifuge at 12,000 x g. then do your A280 scan, SDS gels, with and without reducing agent (DTT). About the dialysis procedure, use only demettallized tubing, or purchase high grade tubing (see Pierce chemicals), decide on the pore size, that depends on the smallest protein you expect to isolate.

Let me know what you get. I could not open your attachment, so can't comment on that.

If you are losing your resuspended protein on the filter, it may be that your protein is not dissolved, or is badly aggregated in the high salt condition. Try centrifuging instead. See whether your protein stays in the supernatant. If not, you might want to try resuspending it in a larger volume to reduce the salt concentration.

The most suitable procedure for desalting will depend on what equipment you have available and the amount of time you are willing to spend.

Dialysis is easy to do, but time consuming, taking at least a day, and uses a large volume of buffer. Your salty sample will probably become diluted during dialysis.

Desalting by gel filtration chromatography can be done quickly as long as you have the column prepared in advance, but your sample will be significantly diluted and you will have to test each column fraction for protein.

If the starting sample volume is small, you can use a gel filtration spin column (a method originated by Harvey Penefsky in the 1970s, I believe). This is a fast method that is simple and quick to set up, avoids dilution, and has good yield if the sample has a high protein concentration. You will need a low speed centrifuge. Pre-packed columns can be purchased (Bio-Rad is one vendor), or you can pack them yourself with Sephadex or Bio-Gel. Sample dilution is avoided because most of the buffer is spun out of the column before the sample is loaded. A second spin elutes the protein. You probably won't get all the salt out with one spin column. Use two in sequence for more thorough desalting.

The method of repeated diluting and reconcentrating by ultrafiltration is another fairly rapid alternative if you have access to either a pressurized ultrafiltration device, or centrifugal ultrafilters.

Do you mean clear or clean? If the solution is literally cloudy, the aggregates may result from lipid or organelle debris; I would see first whether centrifugation can clean it up, and check whether the activity remains in the supernatant-- a microfuge can do this quick test. My favorite gentle way to remove small molecules was dialysis against saturated sucrose, with magnetic stirring. Change the solution if needed, but small diameter tubing can shrink in a few hours (or less, with small volume). Dialysis tubing clips make this a simple, leak-proof procedure.

Apart from the hypotheses stated above by other contributors, there is a possibility that your enzyme requires a cofactor which was lost during filtration. Sometimes holoenzymes with weakly bound cofactors (e.g. FAD) become apoenzymes during purification steps. If this is your case, try adding the cofactor to your buffer.

Good luck!

Thanks for the suggestions, I checked the filter and it was cellulose acetate :-/

I found 0.45 um PVDF filters, but they are pretty small. I'll try them and the centrifugation, that's probably fastest way to do it.

Just for your information, the filtration and centrifugation worked (the activity even increased, probably some interfering impurities were removed), but I lost about 60% of activity during subsequent desalting on HiTrap column.

With dialysis I had most of the activity the other way, so that will be probably my method of choice for next time.

Which is something I don't understand, because if the enzyme lost cofactor as Fernando suggested, it should loose it also during dialysis, right? So why could it loose most of activity during quick desalting?

You can use one of the Amicon Ultra Centrifugal Filter Units. Choose the correct one based on your protein's molecular weight. Here is the one for the proteins with molecular weight 10 kDa and higher:

http://www.millipore.com/catalogue/module/C78144#0

You load your protein sample, add the buffer of interest and centrifuge. Discard supernatant, add the buffer again, centrifuge again. By repeating these steps again and again you will gradually decrease the salt concentration in your sample, while protein concentration (and activity I believe) will not change (if you choose a correct filter as I stated before). It works MUCH faster then dialysis, much better then desalting column and you have much more control over the sample's final volume/concentration. You will only use centrufugation, which you already know doesn't affect the activity of your protein (according to one of your answers).

And remember to check supernatant for the protein presence before you discard it (sometime filters can be damaged, or you may choosen a filter with the wrong cut-off etc.).

I know these filters, I use them regularly. Just yesterday I spend few hours trying to concentrate quite diluted sample. When using it for crude extract, it usually takes days to concentrate.

BTW SUPERnatant is what stays up