xaxax

xaxax, on 02 November 2011 - 01:53 AM, said:

Correct.

xaxax, on 02 November 2011 - 01:53 AM, said:

Correct. (However, be careful with using high pH with proteins, they may not be stable.)

xaxax, on 02 November 2011 - 01:53 AM, said:

Yes, 1 pH unit away from pI could be OK for binding to ion exchanger (go 2 units if you want to be sure).

xaxax, on 02 November 2011 - 01:53 AM, said:

This solution would not have any significantbuffering capacity at pH 10. I would rather try sodium carbonate buffer. However, if you want to use pH gradient elution it may not work properly with carbonate buffer. Gradient elution is quite tricky to perform properly, it requires buffer with wide working range, usually a mix of several (3-7) components instead of usual two. That's why most people use simple salt gradient.

xaxax, on 02 November 2011 - 01:53 AM, said:

Because it is neutral salt (doesn't change pH of buffer).

xaxax, on 02 November 2011 - 01:53 AM, said:

It is not absolutely required but you may want to do this. It would be best to dialyse to buffer in which your protein would be most stable. Phosphate buffered saline (PBS) is quite good.

xaxax, on 02 November 2011 - 01:53 AM, said:

Tris HCl - buffering agent
NaCl - neutral salt, proper ionic strength of solution
Urea - chaotrope, denatures protein, destroys higher order structure of proteins
Guanidine hydrochloride - chaotrope, denatures protein, destroys higher order structure of proteins
Triton - detergent, solubilizes proteins
DTT - reducing agent, destroys disulphide bonds
Na₂HPO₄ - buffering salt
EDTA - chelating agent, binds metal ions
Glutathione - antioxidant, protects proteins from radicals

Wow, that is quite a list of questions.  I will do my best to give you some answers and hopefully others can fill in the blanks.  From the protocol you described, it sounds like you had bacteria overexpress your protein, you broke open or "lysed" you bacteria to harvest the protein and then centrifuged to remove the insoluable cell debris.  The addition of Urea and triton seems like a step to unfold or "denature" the protein, and possibly to separate it from membranes or nucleic acids.  The addition of Guanidine tends to be used to separate protein from nucleic acid, but it can also be used to continue to unfold proteins or separate proteins from complexes.  I'm really not sure why you would add both mercaptoethanol AND DTT to the solution.  These are both reducing agents, which try to mimic the chemical environment inside a cell (which is reducing, compared to a lysate which can be oxidizing).  Basically, the ME and DTT prevent the formation of disulfide bonds that could aggrigate your protein, and depending on the concentrations, they can also be used to break disulfide bonds and help unfold proteins.

The last step in the process is a refolding, although it is not clear exactly how you are doing this.  This can be done by dialysis against a non-denaturing buffer like what you have listed, or possibly on a column where your protein is bound, by slowly changing the concentration of the buffer flowing through the column.

As for your Ion exchange column, typically a salt gradient is used because that is a non-destructive way of eluting protein.  For many people, the protein is still folded normally when they apply their sample to the column, so they don't want the protein to undergo major changes in pH, since that can potentially unfold the protein or inactivate it.  Any salt can be used, but NaCl is typically used in most extraction buffers, so the idea is to keep the chemical composition of your buffer systems consistent from extraction to loading to elution.  I have done ion exchange with KCl and it works just as well, however, potassium is not compatible with SDS-PAGE and will cause preciptitation if you try to analyze your proteins without dialyzing away the potassium.  If you are using NaCl in your extraction buffers, and you will be using SDS-PAGE, just stick with elution using a NaCl gradient.  Depending on the concentration of NaCl that your protein elutes in, you may need to dialyze it, but I would say that anything 200 mM NaCl or below can usually be used without dialysis.  Again, every protein and protocol is different, so you have to use your own judgement depending on your protocol.

Lastly, for the pH of your buffers in doing Ion exchange on your protein with a pI=8.9, I would first consider my resin.  You need to check if your resin can handle buffers of a pH=10 or higher.  Some resins don't react well to pH extremes and you don't want to ruin your column.  Second, you have to consider protein folding.  If you are denaturing your protein in Urea and Guanidine, then it really doesn't matter what pH you are at, but if you are trying to keep it folded, you really shouldn't force it through such extreme pH changes.  Lastly, you should remember that at any pH, there will be proteins that stick to an ion exchange column and those that don't.  If your initial buffers are at pH=7, then your protein will flow through your DEAE column, but a lot of other things will stick to it.  Collecting the flow through will actually be a partial purification because you will be getting rid of everything that stuck to the DEAE column at that pH.  You can then take the flow through and run it over a cation resin, without changing buffers, and your protein will bind.  Then you can do a salt gradient and elute your protein.

I hope that I provided some help, but I also want to restate that every protein is different and many times protein purification is half art and half science.  You have to get to know your protein and your protocol through trial and error.

Best of Luck.