The underlying problem of protease cutting.

: I compare dishes in protein, I have a protein need to do enzyme cutting, protein expression is mainly inclusive, dissolved in 8mol/l urea, or 1%
. SDS
, the protein has been denatured, please ask
protease
cut can be in the denaturation of the conditions of enzymatic cutting, or must wait until after protein complexity can not be enzymatic cutting? Can you do this by passing the denatured protein through dialysis into an enzymatic buffer system, and then enzymatic cutting, so that those denatured urea and SDS are removed, do not know that such operators do not conform to the theory?
: Since your protein is inclusive, why not connect directly to pet21 or pet24, so that the protein is expressed without tag, there is less enzyme cutting this step, isn't it?
Ask: Well, my subject is then my last brother did, there is a 3C protease cut point, expressed membrane protein, purification needs enzyme cutting down, but I have not figured out a concept, urea and other dissolved proteins are denaturing agents, such as TRITON0100 those non-ionized Decontamination agent dissolved protein is a non-denaturation condition, what is the difference and difference between the two proteins, as membrane protein is generally dissolved with TRITON-100, I express hunger this membrane protein insoluble in TRIOTON, and tolerance to SDS or urea, I purify how to deal with enzyme cutting? Please also ask for advice.
: Well, urea and hydrochloric acid are denatured proteins, and if compounding removes them. Descaling agents such as triton x100 or sds are designed to insoluble proteins, such as your membrane proteins, in water.
you can find some articles about membrane protein complexity to look at.
descaling agent, if very strong can also let protein denaturation, but and urea is not the same process.
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