Hi everyone,
Does anyone happen to know the solubility of casein in urea solution?
I have a problem with the purification of recombinant casein as most of the protein are produced in inclusion bodies. Therefore the buffer from resolubilization consists of Sodium Phosphate, NaCl, imidazole, and 8M urea - this is also the binding buffer. The purification is using HisTrap columns. However I find this to be a very high concentration of urea and causing so much trouble during the loading phase, mainly with column overpressure. I want to reduce the urea concentration however not sure which concentration is enough to solubilize the IB. Is 8 M urea really necessary?
Or are there any advice or alternatives for this purification?
This is all new to me so there are lots of aspects that can be optimized.
Thank you in advance.
Dissolution of the inclusion bodies might be better in 6 M guanidine-HCl than in 8 M urea. I would not recommend lowering the denaturant concentration. You should give it a few hours with stirring. You will probably have to slow down the flow rate on the column because of the high density of the solution.
Have you tried modifying your buffer by adding calcium and alkaline conditions...A bit of Triton X-100 additive may also work to form soluble micelles.
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