2 replies to this topic

[MariaG]

Dear all,
I have yet another problem with the protein I am purifying, I have tested the quality and amount of purification on SDS-PAGE followed by coommassie staining, there was a very nice and clear band, it was a month ago. Today I have done SDS-PAGE and coommassie again and the same band of the same protein and the same amount of the protein is barely seen! Therefore I have concluded that the protein was partially degraded. I keep it at 4 C, since it looses activity if frozen, I sterilize it with 0.2 uM filter.
Is there something I can do in order to prevent protein degradation? Add glycerol? Increase salt concentration?
Thank you  very much in advance.

[madrius1]

MariaG, on Mar 18 2009, 06:19 AM, said:

Dear all,
I have yet another problem with the protein I am purifying, I have tested the quality and amount of purification on SDS-PAGE followed by coommassie staining, there was a very nice and clear band, it was a month ago. Today I have done SDS-PAGE and coommassie again and the same band of the same protein and the same amount of the protein is barely seen! Therefore I have concluded that the protein was partially degraded. I keep it at 4 C, since it looses activity if frozen, I sterilize it with 0.2 uM filter.
Is there something I can do in order to prevent protein degradation? Add glycerol? Increase salt concentration?
Thank you  very much in advance.

What about proteases inhitibors?

[mdfenko]

if you can't freeze it then you can try adding glycerol to about 50% and then store at -20C. it won't freeze but should survive longer.