Is there any influence of heat denaturation on outer membrane proteins (bbarrel)? Except defolding of course, something like permanent structure change?
If protein will be expressed as inclusion bodies and then solution will be exposed to heat (15min ~60-70°C), can I still refold it without any problems and the fold will be all right?
Also, already folded protein, exposed to boiling heat and folded again will have any changes in the structure or functionality?
The most common cause of protein folding is the loss of disulfide bonds
Due to oxidation, reduction, and buffer concentration
As for the temperature, I have no known yet
For me urea at above 8.5M got precipitated, probably because of the other salts in the buffer. I agree with Jorg that heat denaturation is not usually a preferred method. I tried denaturing my protein once by heating and just when the buffer hit 50C I noticed precipitation.
@Chandra urea can precipitate maybe because you keep it cold, but here you have to deal with carbamylation of your protein if you will keep it longer not on ice.
I usually use 6-8M urea and its fine for dissolving my IBs completely
@Jörg thanks for answer. So if everything will dissolve in Urea after heat exposure then with proteins is everything fine and they should refold properly? (like those ones without heating?)
No no I am not heating to solubilize the protein. Our French Press is broken and I am using sonicator which is giving a lot of heat to the sample, even in low power and 50/50 s. Plus I was curious what happend with protein after heating.
It is on ice but it still gives a lot of heat. I think its not working properly or the tip is already damaged. I will do refolding tests on Monday, will se how it works.
Thanks for all answers!
sorry, but maybe one of you can help me with my question. I have several GST fusion proteins that are insoluble or they form inclusion bodies. I did use 8M urea to solubilized my proteins, which I did not have problems and then I tried to purify them using the Pierce® GST Spin Purification Kit, but my protein did not bind to the column. why should I do to purify my protein or help to bind to the column? thanks
by the way I diluted my protein 1/8 PBS+0.05 triton x-1oo
I believe you have diluted your inclusion bodies 1:8 in PBS+0.05 triton x-100, if so was the protein solution clear? My understanding is that at Urea concentration of below 2M the protein is not fully denatured which could cause either aggregation or misfolding, and that might affect the tag. I know this is the case with His tag for sure and am hoping the same with GST tag as well.
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