I removed small domains from my enzyme. I want to know which one (truncated or wild-type) is more likely to aggregate at different times at 65 centigrade. Can you give me a strategy to go about doing this?
thank you Dear Arshad
I think it's opposite. truncated enzyme have less possible to aggregate. because I check temperature stability for both of them. truncated enzyme is more stable.
my enzyme is cellulase. yes it's thermozyme.
its small domain is beta sheet structure.
if you want other details, I'll say
thanks a lot Dear Arshad.
I can reach interesting result from your answer.
molecular simulation showed this domain(ig-like) has function in stability. but experiment data is opposite. in your opinion, what is the best way to assay aggregation?
If you are interested in the kinetic of aggregation of you samples, UV 350 nm will help you so much.
It is easy and reliable
Hello Ahmad
yes, kinetic of aggregation is important for me. can you give me a protocol about that? I want to know protocol that I have in mind is correct
TNX
Regards
You can find the method in this paper:
Effect of Arginine on Pre-nucleus Stage of Interferon Beta-1b Aggregation
http://www.ncbi.nlm.nih.gov/pubmed/25142823
Good Luck
After incubating the enzyme at 65°C, you can go for series of experiments like : Turbidity measurements, Rayleigh Scattering, ThT binding, Congored binding, Circular dichroic measurements, ANS binding ,Transmission electron microscopy and Scanning electron microscopy. The all above mentioned methods will provide you deep insight about the aggregation along with change in secondary structure occurring in your enzyme in desired condition. I have performed most of these experiments for my research article.
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