Actual my protein is inclusionbody. so then i decided to solubilize that protein in 2M Urea and I'm also given 2 detergent washes to recover pure protein before solibilization.
Hi, urea according to the theory is not so good denaturant as guanidine hydrochloride and proteins are usually stable in 2M urea. However, everything depends on that what is you protein. Some of them are highly unstable and partial denaturation might be observed in 2M urea. If you are interested in denaturation of your protein you mayAdd your answer increase temperature as well as 2M urea.
- You mention 'degradation' which i assume means hydrolysis. If your sample contains proteases then then protein may be susceptible to degradation. Denaturing agents open up the protein structure and potentially reveal more susceptible bonds for cleavage. 2M urea is not a high concentration but the primary consideration is whether you have proteases in the sample. if you have no proteases (unlikely) you will have no degradation. If you have proteases the sample will probably degrade more if the danaturant concentration is increased. You can add protease inhibitors if required.
THANK YOU SIR,
I didnt add any proteases but there may be any proteases.
Artur Krężel sir i did same work before also,but that time nothing happened, and i have recovered cleared denatured protein for refolding
Dear Srikanth:
Usually 2M Urea works fine to solubilised the inclusion bodies if your over-expressed protein is from a mesophile. If your protein contains high amount of amino acidic residues, you may increase urea up to 4M to get fully solubilised inclusion bodies. I would suggest you to avoid the use of proteases.
Good luck,
Rosa María Martínez-Espinosa
Sounds like you have protease contamination if your protein is degrading in 2M urea.
Add protease inhibitors.
thank you
Deepan Shah
Rosa María Martínez-Espinosa
Nick Gee
Artur Krężel
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry