I would suggest to replace your phosphate buffer with Ammonium Bicarbonate buffer or water during dialysis.

The volume increase of the sample was due to the sample having a higher osmolarity than the dialysis buffer.

I would guess that the crystals are ammonium phosphate, although this is not consistent with the high aqueous solubility of that salt. If your sample had a lot of magnesium or calcium, the crystals could be the phosphate salts of those ions. In any case, as Viktor Butnev said, a different dialysis buffer is warranted to avoid the crystallization. Many other buffers are possible. Ammonium bicarbonate is useful for mass spectrometry, or when the sample is to be lyophilized, because it is volatile. Tris or HEPES are often used for general purposes.

Since you are not fractionating by ammonium sulfate precipitation, maybe you should just skip this step.

part of your problem may be due to the length of time you did dialysis and the stability of your protein. It took you a week to dialyze. I would use a desalting column (spin column) from thermoscientific. You just add the protein to the top, and spin. You can equilibrate the column in any buffer you want. I believe they come in either PBS or Tris. I would use the tris buffer per recommendations from Adam to avoid phosphate buffer.

I would strongly advice against dialyzing for a week (as M. Moss also discusses).

Desalting (spin) columns usually work very good.

See also: Ibrahim et a., (1990) "Preparation and purification of Amylase enzyme produced by Streptomyces aureofaciens 77"

Arch. Pharm. Res. 13(1), pages 28-32.

Were you dialyzing the amylase preparation under cold (2-8C) conditions? If the phosphate buffer was prepared at room temperature, it could recrystallize, if it was at a sufficiently high concentration to begin with.

I have seen this issue when I've prepared concentrated sodium phosphate stock solutions, then stored them under refrigeration.

I'm wondering, do you required to increased your ammonium sulphate concentration till 100%? Usually, it works brilliantly at about 60 - 80%.

As for your protein precipitate, you may want to increase the volume of the phosphate buffer (concentration between 10 - 50 mM) from 500 ul to 2 ml for maximize the solubility of your protein.

Try to dialyzes your protein sample with 5 mM phosphate buffer at low temperature ( 4 - 10oC) and change the buffer in every 4 hours interval. Change for 2 - 3 times, your protein should be fine and it will clear out most of the salt.

As for the large volume of your protein sample, you may want to use the protein concentrator to concentrate your protein sample.

Thank you!

I have used this protocol (ammonium sulphate precipitation) for a long time (more than 10 years) and all I cay say is that ammonium sulphate precipitation and phosphate buffer is not the best combination. I suggest you to replace phosphate buffer by Tris or Hepes as Dr. Shapiro suggested before. 

If you don't have data about which is the ammonium sulphate concentration that precipitates your protein, you should start by using non linear increasing ammonium precipitation. 

What was the source of your protein? Did you check the supernatant after salting out for remaining activity? It may be that your protein stayed in the solution. That happened to me as well.

Ammonium Sulphate precipitation is usually used to recover proteins from a large volume to make sample handling easier.   For example, if you have got a secreted protein which is present in litres of growth buffer, then you use ammonium sulphate to precipitate the protein and resuspend it in a smaller volume to make it easier to perform purification using gel filtration or other types of chromatography.   After ammonium sulphate precipitation, it is usually impossible to recover all the precipitated protein in a soluble form.   It is common to perform a gel filtration step after the precipitation/resolubilization step to remove salts.   Phosphate buffer is not a good buffer to use in any purification step because of the low solubility of metal phosphates etc.   Tris buffer especially at pHs 7.0 to 8.0 works best.

If you have to use ammonium sulphate precipitation, you must check the % saturation of ammonium sulphate at which you get maximum precipitation/resolubilization of your desired protein.   This might mean doing several trial experiments to check this out.    

First You can replace the PBS with biocarboante buffer and dialyze the small amount of protein to optimize the optimum percentage of ammonuim sulfate. The experiment time can also be shortened for optimization of Cut percentage of ammonium sulfate. Hope it will help.

the protein is microbial protein.....can ethanol precipitation be carried out for this protein?.... if yes then what are the advantages of this method over ammonium precipitation method. i need to concentrate and purify the protein for further study like characterization based on temp and ph.....

Most proteins are denatured by organic solvents like ethanol, so this method of precipitation would not be desirable if you want to characterize the structure and function of the protein.

Hi Himanshu,

Instead of using ethanol you can try acetone for the precipitation of your protein. Yes it is right thatmost of the proteins are sensitive towards organic solvents like ethanol. You can carry out the process in -20oC conditions and use the solvent precooled for overnight.. Hope it will help.

Good luck

So what about some large-scale chromatography like ionex or hydrophobic chromatography for the capture step? That usually works best.

i used ethanol and got good precipitates but now the precipitates are not completely soluble in sodium phosphate buffer but partially soluble.....i carried out enzyme assay and protein estimation  with this sample.both are giving good absorbance........

now my question is do i need to go for the dialysis of these precipitates? or can i directly proceed for gel chromatography?

You can probably proceed directly to gel filtration chromatography. Your protein may elute as a mixture of monomers (eluting late) and aggregates (eluting early) if some of the protein was aggregated by the ethanol precipitation.

Hello Adam

thank you so much for giving a go ahead with gel filtration chromatography....

i tried dialysis of the protein precipitates isolated by ethanol precipitation protocol but when i checked its enzyme activity and protein estimation it was less then the samples without dialysis.. so i guess i should skip this step of  dialysis.