Hello! I am precipitating some proteins (with either acetone or ammonium sulphate) and I need to resuspend the protein pellet in a buffer that properly keeps the proteins soluble AND which is compatible with cellular stimulation (i.e, does not contain compounds that are super toxic to cells). Of course the protein extracts will be diluted in cell medium for the stimulation and therefore the presumably toxic compund(s) used for protein solubilization will be diluted, but still I want to make sure I am not using something that will kill the cells at a nanomolar level.
Thanks a lot for your help!
For protein precipitated with ammonium sulfate, you can remove most of the ammonium sulfate by centrifuging the suspension. The pellet can be dissolved in growth medium or whatever buffer you like, such as PBS. It will still contain some ammonium sulfate, however, which may be undesirable. You can remove it by passing the protein solution through a gel filtration spin column equilibrated with the same buffer. To make sure the protein solution is sterile, you can pass it through a sterile syringe filter. Finally, measure the protein concentration.
This may also work with acetone-precipitated protein, but I would rather use ammonium sulfate-precipitated protein. It's more likely to retain the native structure.
Thanks a lot for your answer! Indeed, after resuspending my protein pellet I am desalting the protein solution to remove any residual AS or acetone. However, the problem is that I need to use stringent detergents to get all the proteins out, so the proteins denature and after acetone or AS precipitation I get a protein pellet that is almost impossible to dissolve because proteins are aggregated and form clumps. I have tried to sonicate and it does not help. So, I need a buffer to resuspend my protein pellet after precipitation that ensures solubilization of hydrophobic proteins (to prevent them from aggregating one to another) that is not toxic to cells. Thanks!
I would rather work on the first step. It's very likely that you denature your protein, especially with acetone; you can't solubilize it again afterwards. Depending on how much solution you have, the MW of the protein and at which contrentation, you might concentrate in a centricon type filter and then wash directly with your buffer.
If your protein requires strong detergents to extract, then it must be a membrane protein or other insoluble protein. A membrane protein will not be soluble unless you include detergent or a strong chaotrope such as urea or guanidine.
One strategy would be to try to find a non-denaturing detergent that will extract the protein. Another strategy would be to dissolve the precipitated protein in 6-8 M guanidine HCl, then gradually replace the denaturant with a detergent that will keep the protein in solution, and perhaps allow it to refold, at least partially.
At best, you will end up with a solution of the protein in detergent. This is unlikely to be compatible with cells. You might be able to reconstitute the detergent-solubilized protein into nanodiscs or proteoliposomes that could be added to cell cultures.
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