I am trying to purify the beta sub unit of the Rhodococcus proteasome.I am using denaturing purification with 8M urea.The protein falls out of solution immediately after purification. overnight dialysis withTEV makes the protein disappear completely.I have also checked solubility in presence of detergents and DTT but even they have been of little help.Can anyone suggest any particular protocol to purify this protein or a trick to get it into solution and make it stay there.
Is it possible that once the urea is dialyzed out the protein refolds and digests itself? If so, is there a reversible inhibitor that can be included in order to prevent this?
I tested the autocleaving hypothesis but seems it is a solubility issue.I need to figure out a way to keep it soluble in absence of urea.
You said you already tried detergents. Which ones did you try and at what concentrations? There are many detergents with a variety of properties.
I have tried triton X100, SDS and tween 20 @ 0.1 & 1% .but my usage of detergents is restricted with respect the future experiments.Are there buffers that help increase solubility.I am currently using HEPES
pH, salt concentration, and temperature can influence solubility. As for detergents, if you are willing to try them, I would suggest trying n-octylglucoside, dodecylmaltoside, CHAPS, and Zwittergent 3-12 and 3-14. Some sugars can be helpful at high concentrations, such as glycerol, sucrose and trehalose.
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