do i need to desalt my protein after purification - (Apr/02/2008 )
i've manage to purify my protein using the ni-nta column.i got 1 clear band on my sds-page.rite now, i want to use my purified protein for enzymatic studies.
i just want to know, do i really have to desalt my protein first before proceed to the cleavage session..does the salt contain in my buffer during purification disturb the cleavage enzyme for his tag removal?if not, i want to use it directly in the his tag removal step.
another question i want to ask, what is exactly influence the protein solubility and the intracellular location when it is expressed in the e.coli ?is it becoz of the signal peptide, the nature of the protein or depends on the host cell?i just want to know how to determine the protein is soluble in the cytoplasm, secreted into the periplasmic space or located in cytoplasmic inclusion bodies.
i just want to know, do i really have to desalt my protein first before proceed to the cleavage session..does the salt contain in my buffer during purification disturb the cleavage enzyme for his tag removal?if not, i want to use it directly in the his tag removal step.
What is the buffer for your cleavage enzyme? If it's close to your elution buffer (apart from the imidazole), it should be OK to use it. Why don't you try a test digestion: desalt an aliquot of the eluate, using the correct buffer for your protease. Test that against some untreated eluate and see what differences you get in digestion. There's nothing quite like doing an experiment to know what to do next. (Also, we might be able to give a better answer if we knew what enzyme you're wanting to use, and the preferred conditions
There are protocols to prepare periplasm, as well as the usual total cell lysate. Sorry to be less than helpful, but I'd suggest you Google periplasmic fraction purification and see what comes up.
As for the question of solubility, there are a large number of known variables, and quite possibly, a few unknown variables. Induction temp, concentration of inducer, promoter strength, availability of required tRNAs (BIG are of debate here) are just a few. There are also a number of sizeable reviews, often aimed at the structural genomics field (but completely applicable to smaller scale projects) that can give you a handle on where to start. Off the top of my head, try reducing the temperature of induction and the concentration of inducer.