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I have a protein with a theoretical $\mathrm{pI}~9.0$ currently stored in $\pu{25 mM}$ tris and $\pu{250 mM}$ $\ce{NaCl}$ $(\mathrm{pH}~8)$ at $\pu{−80 °C}.$ I need to do experiments in HEPES at $\mathrm{pH}~7.2$ and what I did previously was to just freshly buffer exchange my protein from tris to HEPES using Amicon® molecular weight cutoff filters and then use it either on the same day or 1–2 days after, storing the protein solution in my desired HEPES buffer at $\pu{4 °C}$ for that time.

Will my protein degrade when it is in HEPES at $\mathrm{pH}~7.2?$ How long can I store it in HEPES buffer at $\pu{4 °C}$ before it degrades? Can I freeze my protein in HEPES buffer for a short period of time? Is PAGE the best way to check for potential protein degradation?

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    $\begingroup$ What's the protein concentration (also useful to consider is MW, type of protein, construct, etc)? Why do you expect degradation? How pure is it? Did you or can you add any protease inhibitor (or cocktail)? Do you maybe mean aggregate rather than degrade? Aggregation is more likely at higher concentration. Aggregation depends very much on the particular protein. Lowering the pH relative to pI should reduce chances of that occurring but proteins being proteins.... $\endgroup$
    – Buck Thorn ♦
    Commented Apr 15 at 11:14
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    $\begingroup$ MW is 26 kDa. Purity is >95%. I cannot add protease inhibitor. I would not expect such a fast degradation as I could show good binding to my target but I just want to avoid falling into a trap of misinterpreting my data. But I am correct in the assumption that longer term storage of the protein in HEPES is not good since the pH is way off from the pI? $\endgroup$
    – TheChemist
    Commented Apr 15 at 11:27
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    $\begingroup$ I would think lower pH would be better for a high pI protein - farther away from pI means more ionized, less liable to aggregate. A before/after comparison of 1D NMR and/or CD should be useful (not sure if HEPES is compatible with CD though). What's best depends somewhat on how much sample and concentration you have. $\endgroup$
    – Buck Thorn ♦
    Commented Apr 15 at 11:36
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    $\begingroup$ Hm ok. I only know this rule of thumb that for protein storage the pH of the buffer should be within 1.0 unit of the pI of the protein. And HEPES only buffers to a maximum of pH 8.0 and Tris up to 9.0 which I think is why its common choice for prolonged storage. I will do some testing. Thx for the suggestion. $\endgroup$
    – TheChemist
    Commented Apr 15 at 13:48
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    $\begingroup$ Could be we are talking about different issues regarding pH. One is the ideal buffer pH value during purification of the protein. $\endgroup$
    – Buck Thorn ♦
    Commented Apr 15 at 14:25

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