Was the pH of the solution the same in all cases? High concentration of (NH4)2SO4 might have changed the pH of the solution that can maintain proteins in their soluble forms. You need to keep all parameters constant to reveal weather your observation is the result of change in concentration of ammonium sulphate or not.

Hi Pichia. Precipitation using Ammonium Sulfate is protein dependent, not Amm. Sulf. dependent. Some proteins precipitate in low amm. sulf. but remain soluble in high amm. sulf.

It all depends on the hydrophobic surfaces on proteins. Low amm. sulf. forces the hydrophobic surfaces of some proteins to combine and precipitate. High amm. sulf. can work as a salt that coats the proteins and repels interactions.

Hi!

It is actually quite unusual as it sounds. But if you are observing it, there must be a definite reason behind. Once if you can try adding from 0 to 20% salt and gradually increase it to 60 by increasing concentration bit by bit, would you expect salting out first and then solubilisation again? Based on your description, I can say that the structure of your protein (the percentage of hydrophobic and hydrophilic patches that it has) would determine the right ammonium sulfate concentration required for it to be dissolved or precipitated. It might so happen that your protein has quite a large percentage of hydrophilic amino acid residues which easily gets destructured when water of solvation is taken up by the salts (as low as 20%). Then when the salt concentration is increased (from 20% to 60%) the excess salt molecules cover up the hydrophilic patches (instead of water of solvation around them now), and maintains the protein in soluble form. It would solely be determined by the protein structure.

Hope it helps!

I agree with Alemu that the pH of the solution will change on increasing addition of ammonium sulphate and also worry about adding solid salt. there may be a risk of the salt dissolving and locally having a very high concentration which precipitates protein which may not redissolve when the whole reaction is mixed and equilibrates. The pH can be stabilised with 50mM HEPES to investigate its effect.The attached is quite useful,

Article Protein Precipitation Using Ammonium Sulfate

Hi again. To address Paul's and Alemu's concern, I would suggest you to follow something which we do and it works fine without any such complications as mentioned above. The protein is always in a buffer which would maintain a pH likeable for the protein (which will keep it stable). We add the protein solution to be concentrated, in a small beaker with a small magnetic bead, put the beaker in a ice bucket before adding ammonium sulfate to it. As you go on adding the salt the stirrer ensures proper mixing and homogeneous effect of the salt on your protein. This entire set up can be done in cold room, where temperature is not more than 4 degree to prevent any degradation of your protein. Hope it helps!

I guess your protein has very high molecular weight or if it has low molecular weight then it might be forming higher oligomers in solution to make it very big molecular machinery. In this scenario, 30 percent glycerol in the buffer will help you to avoid precipitation at 20%. Additionally, i dont have idea about the buffer composition but it should have 250mM KCL which also avoids precipitation. Why 20 % precipitation is not fine with u? I mean why specifically you want 40 or 60 % cut?

It's not that it is not fine with me.

I am just trying to understand the behaviour of a protein that precipitates at lower ammonium sulphate rather than higher.

I think oligemeric structures could make sense but it still odd. I know it is not forming oligomers in my buffer as I did an analytical size exclusion but it could be forming oligomers with the addition of salt. It would be odd that it would salt out at 20% and then as I raise the salt to 40% it salts back in.

Hi Pichia. Ammonium sulfate protein precipitation has nothing to do with their size, structure or function.

This will be more clear if you have worked with hydrophobic and hydrophilic peptides. Hydrophobic peptides are not soluble in aqueous solutions and will precipitate because their hydrophobic residues prefer to stick together rather than be exposed to the hydrophilic solvent. It is more energetically favorable for hydrophobic surfaces to stick together.

The same thing happens with proteins. What matters is that the AS makes the solution very polar. In a very polar solution hydrophobic patches of proteins stick together and become insoluble in this environment.

It is odd if a protein salts out at 20% AS and salts in at 40% AS. I have never experienced that.

The only reasons I can think of is if it was an oligomer that salted out at 20%, dissociates at higher AS% or is denatured.