Try centrifuging the cloudy suspension to pellet any aggregated material before loading the supernatant onto the column.

Try centrifuging the cloudy suspension to pellet any aggregated material before loading the supernatant onto the column.

The precipitate that remains undissolved could be aggregated protein from the initial precipitation. Are you cooling the broth before adding the acetone? what is the ratio of acetone to broth?

One option would be to dilute your sample since a high protein concentration leads to precipitate as well. Another is to check the pI of your Protein and compare it to your adjusted pH. Third option could be another preciptation method like TCA.

Did you have check the total amount of protein was precipitated and the volumn. For example, if you have 100 mg of protein in 100 ml, and after precipation you ressuspended all precipited protein in 10 mL of buffer, you are saturating the solution. When I make protein precipitation, I ressuspend all precipitated protein in 1/3 of initial volume of sample. 

All very good suggestions -

Adam's suggestion gets you to your end point sooner - over the column.

When you re-dissolved the ppt. in the phosphate buffer, was that hazy too or was the haziness a result of the refrigerated storage? 

Amended on 1 January 2017

This is an important question.   Famous American Agnostic Dr. Irving Langmuir (inventor of the term "pathological science") has already suggested that the aggregation (and subsequent precipitation) is irreversal in chemistry, but it is also true in the biochemistry.  

As Italian Dr. Claudio De Felice and me have suggested, the aggregation is irreversal even in non-ionic detergent, but aggregation is dispersed only after the boiling with SDS; i.e., aggregation occurs mainly by hydrophobic interaction (please see file; CA agg Dr. De Felice).  

Since the aggregation article has been published by us, I must have to explain why protein aggregation has occurred at -20 degree Celsius (°C)/absolute temperature at 253 °K, and has not occurred at -80 degree Celsius (°C)/absolute temperature at 193 °K and liquid nitrogen of -196 degree Celsius (°C)/absolute temperature at 77 °K.

Hydrophobic interaction due to the Van der Waals force (mainly due to dipole-dipole interaction) is active at 253 °K, and distance among protein moecules is closely fixed at high concentration (Van der Waals force acts in accordance with the molecular distance of 1/10-6 ).   Famous Dutsch Dr. Johannes Diderik van der Waals (Universiteit van Amsterdam, Amsterdam, The Netherlands) has indicated that the hydrophobic interaction increases in accordance with molecular weight, which suggests protein separation by reversed-phase HPLC becomes difficult for large proteins.   Thus, I have used low temperature of 15 °C (RP-HPLC) or 8 °C (HPLC-Soap-SEC) for protein analysis (please see file; Lysozyme by RP-HPLC).   Then, I must now consider that more lower temperature is necessary for RP-HPLC-protein determination by using ultra-high-pressure liquid chromatography (UHPLC) pump (upto 1,000 kg/cm2 or 100 MPa may be possible) and possible column-cooling apparatus.

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When 50% (v/v) ethylene glycol or 50% (v/v) glycerol is present, distances among protein moecules are not fixed, and the protein aggregation does not occur even at 253 °K.    

But, the dipole-dipole motion becomes slow at 193 °K and at 77 °K, and the hydrophobic interaction or Van der Waals force then decreases at 193 °K and at 77 °K.   Therefore, protein aggregation at high concentrations of protein molecules occurs only at -20 degree Celsius (°C)/absolute temperature at 253 °K.   

Thus, we have performed the dispersion by non-ionic detergent of the brain membrans and the direct absorption of contaminant proteins to CM-cellulose gells.

The filtrate containing lipoamidase has then been firstly separeted by using DEAE-cellulose column (please see the file; Purify Brain Lipoamidase).    

For another chromatography steps, the concentration by using an ultrafiltration membrane (PM-10, Amicon Co., Danvers, MA, USA) has been chosen.   Other usual concentration methods using aggregation phenomenon (suc as ammonium sulphate precipitation method etc.) have not been used.  

However, absorption of proteins to PM-10 cellulose membrane has been seemed to be considerable, I have been used the concentration method by using dry-PEG20,000 and dialysis membrane tube (made of cellulose).   But the concentrated fluid becomes very viscous.   Then, concentration by cellulose-dialysis tube and dry sucrose or NaCl may become another choice.   Dialysis tube made of agarose may be desirable, although such a membrane is not available yet.   This concentration method requires to see the end time of concentration. 

Another possible concentration method for hydrophobic (lipophilic; LipPhil) membrane proteins may be (1) gel-free IEF by Rotofor (BioRad) with non-ionic detergent and several amino acids instead of high cost Ampholine and (2) sucrose-density ultracentrifugation (please see file; Dr. Kusaka Membrane).

Nice question. you can collect the fermentation broth and directly add ammonium sulphate range from 20 to 65 % saturation, then go for dialysis and DEAE puification. you can also go for ultrafiltaration membrane of particular mol. weight cut off for protein purification. it is available in merck millipore. then elute protein using deae with different gradient using NaCl if you need high purity.

Most of the answers above are relevant.. BUT YOU HAVE NOT SPECIFIED THE ENZYME OF INTEREST... is it membrane bound? cytoplasmic? There are different solutions to your questions. I congratulate you that an acetone ppt shows enhanced activity. Proceed cautiously.  If it was mine I'd dialyse a sample in your phosphate buffer o/n and filtrate, then test enzymic activity. If it drops chances are the protein is hydrophobic then you could consider another buffer (eg CHAPS). Just a suggestion.