Hi Tatjana,

Are you working with purified protein? I usually ignore the instability index in ProtParam. My protein was predicted as unstable, but it remained active after being stored at -20C  for years (with glycerol, of course). As a rule of thumb, don't store your protein samples at 4C if you want to keep them for more than a week. I store my protein in -80C whenever possible. I either snap freeze them in PCR tubes, or in bigger volumes with 10-30% glycerol.

In fact, as my PhD supervisor has told me, never leave protein at room temperature after cell lysis. Keep your protein on ice at all time during purification. If you want to be extra careful, add protease inhibitor to your samples.

If I were you, I would test if it's really degradation by leaving a portion at 4C. Take a small sample every day, boil in SDS buffer and store in the freezer. After a week or so, run them all on a single gel to see if the smaller bands increase in intensity relative to your target protein.

Best regards,

Arthur

Hi Tatjana,

Are you working with purified protein? I usually ignore the instability index in ProtParam. My protein was predicted as unstable, but it remained active after being stored at -20C  for years (with glycerol, of course). As a rule of thumb, don't store your protein samples at 4C if you want to keep them for more than a week. I store my protein in -80C whenever possible. I either snap freeze them in PCR tubes, or in bigger volumes with 10-30% glycerol.

In fact, as my PhD supervisor has told me, never leave protein at room temperature after cell lysis. Keep your protein on ice at all time during purification. If you want to be extra careful, add protease inhibitor to your samples.

If I were you, I would test if it's really degradation by leaving a portion at 4C. Take a small sample every day, boil in SDS buffer and store in the freezer. After a week or so, run them all on a single gel to see if the smaller bands increase in intensity relative to your target protein.

Best regards,

Arthur

 Hi Tatjana,

Arthur is dead right. Any prediction is not real until experimentally proven. As he correctly pointed out, bioinformatics based predictions should not be taken as valid for individual problems/proteins. On a scale of thousands of proteins the predictions may be somewhat correct for a majority of the cases. But it may be completely misleading for a particular problem. Good luck

Sujay

Thank you so much, I will try to track the degradation-process over a week and maybe link it to the ProtParam-value. That is a really good idea!

You helped me a lot!