I do western blotting with these lysates and i just want to minimize the degradation of protein as much as possible and i also want to know whether the storing temperature (-20 or -80) have any effect on the protein integrity.
I would like to add that protein lysates from cells after boiling with Laemmli's sample buffer can be stored safely at either -20 or -80 degree C. The boiling inactivates proteases that degrade proteins. However, certain chromatin binding proteins may require proper processing of the frozen protein samples (a quick thawing of samples at 37 degree C, vortexing, heating, vortexing, and centrifugation) before analyzing by immunoblotting.
That all depends on the frequence you use. If u use it very often, it is better for you to store you lysates in -20, since it will not take so long time to dissolve the lysates. Besides, the repeated freezing and thawing is not good for the protein integrity. U can chose -80 for storing, if u just use it once or twice a month.
I would like to add that protein lysates from cells after boiling with Laemmli's sample buffer can be stored safely at either -20 or -80 degree C. The boiling inactivates proteases that degrade proteins. However, certain chromatin binding proteins may require proper processing of the frozen protein samples (a quick thawing of samples at 37 degree C, vortexing, heating, vortexing, and centrifugation) before analyzing by immunoblotting.
u can store them for long term by quick-freezing followed by storage at -20°C
We routinely store at -80 and use often thawing them in ice. It worked for our proteins including phosphorylated proteins.
I suggest Lysate + inhibitors in -80 as aliquots. Once thawed store in -20 try avoiding repeated thaw cycle.
Hello khushal
I advise you to store the protein extraction in sample buffer as aliquots at -80ºC for minimize the action of proteases although your buffer have proteases inhibitors.
Regards
Boil it in Laemmli buffer, aliquote them and store at -80C. This way you can minimize or prevent degradation. Make enough number of aliquotes in case you are storing without boiling. Repeated thawing can excessively degrade the proteins.
before boiling them at -80. after boiling with sample buffer at -20.
Good Luck!
As others have said, I have been storing lysates with sample buffer after boilong in -20°C and it has been working fine.
Now I have a question and maybe it could be addressed here:
I have extracted proteins both in RIPA buffer and in a more stringent buffer (1% SDS), both with protease and phosphatase inhibitors. I'm running native gels and hence adding a native sample buffer (no SDS) to the lysates.
Should the samples (after adding the native sample buffer) be stores at -80 or is it -20 ok?
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