Dear Miss Merlaen, I studied this problem, and verified that is possible to storage aqueous solution of ascorbic acid. See the paper: Use of column with modified silica for interfering retention in a FIA spectrophotometric method for direct determination of vitamin C in medicine. You can download in my profile. This study was made in pH 5.0 or 5.6. Storing by one hour, at -10ºC and without oxygen the degradation is zero, but in the presence of oxygen it was more or less 1%. But in the pH of study, there is monoascorbate ion, which is very unstable. I suggest to storage at pH

You can store concentrated ascorbic acid in the freezer (-20 ºC) provided it is at low pH (4 or less). It may oxidize slowly due to oxygen and trace metals in the solution so you an store it under argon and/or treat the solution with a chelating resin before storing. Additionally, you can easily measure total ascorbate concentration by reduction of DCPIP so you can always monitor your actual concentration.

In the laboratory there are often cylinders of pure nitrogen. Nitrogen can be bubbled slowly for a few hours  in the solvent that you use to prepare the solution. This eliminates the oxygen present in the water. It is probably the quickest and least expensive procedure. If the pH is acidic it is better.

Make it up fresh when you need it. I wasn't aware that ascorbic acid is a protease inhibitor. What class of protease? Can you provide a reference, please.

I included ascorbic acid in my homogenization medium because I saw it was also included in other papers describing plasma membrane extraction, like "A fruit-specific plasma membrane aquaporin subtype PIP1;1 is regulated during strawberry" (Fragaria x ananassa) fruit ripening by Mut et al 2008)

It's propbably not a protease inhibitor in intself, but it may help to avoid or lower protein breakdown during extraction. This paper (Han, S., Li, Y. Y., & Chan, B. P. (2015). Protease inhibitors enhance extracellular collagen fibril deposition in human mesenchymal stem cells. Stem Cell Research & Therapy, 6, 197. http://doi.org/10.1186/s13287-015-0191-1) shows that it works synergistically with protease inhibitors. 

Ascorbic acid is added to lysis buffers as an antioxidant. The paper on synergy with protease inhibitors is on cellular collagen expression and its extracellular deposition. No interaction between ascorbic acid and protease inhibitors is implied. You just get more collagen deposited in a cellular system by stimulating its expression with ascorbic acid (a well known effect) and preventing its degradation with protease inhibitors. Nothing there suggests that ascorbic acid acts to improve protease inhibitor activity in a cellular or cell-free system. Too bad, I'm always on the lookout for better protease inhibitors.

I'm sorry to have given you false hope. Thank you for the responses!

Maybe you can also help me out with this:  I flash froze the membrane extracts immediately after extraction and stored them at -80°C. Now I want to process them: DNAse/RNAse treatment and ammonium acetate precipitation (which is necessary because the extract is not concentrated enough). Can I pauze the protocol after the precipitation and start SDS-PAGE the next day? If yes, would it be best to store the dry pellet (at 4°C?) or to resuspend the plellet and store like this (at 4°C?). Storage for 24h max.

thank you in advance!

First, ammonium sulfate is the salt of choice for precipitation of protein, not ammonium acetate. Second, if I were you, I would do the extraction in a smaller volume to get the concentration that I need, rather than do precipitation, unless it is absolutely unavoidable. Third, especially for membrane proteins, some people prefer acetone precipitation or TCA precipitation followed by an acetone wash (and some drying), because the pellets can be directly solubilized and run on a gel without dialysis (see below). Have you considered this?

You can keep an ammonium sulfate precipitate as a wet pellet or slurry at 4°C overnight with no problem (you can probably keep it for months like this!). You should avoid drying the pellet as this can cause refractory aggregation of some proteins, especially membrane proteins. You cannot run a resolubilized ammonium sulfate pellet directly on SDS-PAGE because of the high salt concentration. You will need to dialyze or do buffer exchange on e.g. Sephadex G-25 (microdialysis and microspin G-25 columns are commercially available, or you can improvise). It would be best to go from extraction to gel in the same day if possible, but you can pause as you have suggested. It depends on what you want to accomplish.