a few questions about ion exchange chromatography - (Nov/02/2011 )

Hi friends
I am a chemical engineer and am told to purify a protein from a protein mixture. AND I know a little information about proteins. The isoelectric point of the desired protein is 8.91. I know if the buffer pH is above protein's isoelectric point, protein is charged negative. If it is below the isoelectric point, protein will be positively charged.

* If I use a buffer with pH=10 and a weak anion exchanger (DEAE-I have only anion exchanger) and then, decreasing the buffer pH gradually to slightly below the isoelectric point, can i take the desired protein? OR if use a cation exchanger, the buffer pH=8 is enough?
*** What should the content of the buffer be? for example, 5mM Na2HPO4, 1mM EDTA, 100mM NaCl and to obtain a pH=10, i will add NaOH to buffer solution???

Before the ion exchange chromatography, this procedure had been done by others and I think there are some unconscious addings of chemicals. I do not know the terminology of this type of science. excuse me. :-(
1. The desired protein was produced by bacteriums. Then, those bacteriums were put to -80 C.
2. Bacteriums were exploded with osmosis and then, centrifuged. supernatant was taken.
3. Urea, tris HCI, NaCl, triton solution (pH=8) was added to the supernatant and sonicated, then, centrifuged. this was done twice.
4. Guanidin HCl, NaCl, Mercaptoethanol, tris HCl solution (pH=8) was added to the supernatant and centrifuged.
5. DTT (Dithioeritrotriol) was added to the supernatant.
6. The protein was refolded by Na2HPO4, EDTA, Glutatyon solution (pH=8).

* It is known that increase in salt gradient releases the bounded proteins from ion exchange resins. why NaCl is used? Should it be dialysed? what should the dialysis buffer content be?
** What is the aim of these chemicals used in these steps?
All your advices are very crucial for me. Thank you very much. I am waiting for your help.

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.

xaxax on Wed Nov 2 09:53:05 2011 said:

Correct.

xaxax on Wed Nov 2 09:53:05 2011 said:

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

xaxax on Wed Nov 2 09:53:05 2011 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 Wed Nov 2 09:53:05 2011 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 Wed Nov 2 09:53:05 2011 said:

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

xaxax on Wed Nov 2 09:53:05 2011 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 Wed Nov 2 09:53:05 2011 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

I want to thank very very much for your deep interest. You gave me very important notes.
I want to learn the meaning of STABILITY OF PROTEIN at a specific pH. if the protein is not stable at that pH, what occurs?
Ethanol amine or pipperazine have a pH between 9-10. i can not use them for buffer, since they are toxic. However, is there a buffer that has a pH value near 10? and if i use my buffer explained above, does adding NaOH affect to anything? Is it proper to use HCl in order to decrease the pH of mobile phase and the column?

If i can not a good result, i will carry out the procedure with a cation exchanger and a pH buffer 7.5 or 8.

Excuse me. today, I made anion exchange chromatography (DEAE) from the pH=10.
First, I equlibriated my column with buffer to pH=10. Then, I took flow through and wash with a buffer (pH=10). After that, I added a buffer (pH=10) and eluted. Then, i added a buffer (pH=9.5) and when I looked the pH of the column, it was still 10. Although, i added buffers 9- 8.5-8 respectively, the pH was 10 again.
I think there was a wrong with the pH and I decided to make this procedure.
1. equlibriating the column with buffer to pH=10
2. taking flow through with pH=10
3. adjusting the column to pH=9.5 and making a buffer (pH=9.5). then, taking eluat.
4. adjusting the column to pH=9 and making a buffer (pH=9). then, taking eluat.
5. adjusting the column to pH=8.5 and making a buffer (pH=8.5). then, taking eluat.
6. adjusting the column to pH=8 and making a buffer (pH=8). then, taking eluat.
can this be a right way for purifying my protein???
5mM Na2HPO4, 1mM EDTA, 100mM NaCl and to obtain a pH=10, i will add NaOH to obtain the desired buffer solution...

adjusting the pH and then adding buffer, with protein already bound, may be too harsh. generally you would use buffer to change the pH. this takes time and volume because counter-ions are bound to the matrix and have to be displaced.

i'm attaching the ion exchange and chromatofocusing handbook (ge healthcare) in case you don't have it.

thanks for your document MDFENKO. it is very useful.
I want to change the pH of the column from 10 to 9.
the starting pH of the DEAE column is 10. i want to decrease the pH to 9 with buffer. but i can not change it. what should be done?
should i wait for a long time? or should i wash the column 5-10 times with buffer?

see page 46 of the handbook. it gives information and cautions about pH elution.

and, yes, you will have to use a larger volume of buffer to see the change in pH.

Thanks very much for your deep interest MDFENKO, again. I got a lot of information from you.
I am continuing to ask questions and i hope i do not make you bored. Excuse me :-) You know that these questions are very basic and vital and vital questions and, they can be solved by experience.

Now, I found a Sartorius Vivapure S Maxi H column that is a strong cation exchanger and approximate pKa of ionc group is 1.
pI of my protein is 8.91.
I will keep the column in water for 2-3 hours to swell. After I equilibrate the column with 5ml buffer, I will apply my protein solution (10ml) to the column in three portions and, after every load i will centrifuge the column.
After this step, i cannot decide what i will do. I want to make this procedure. Does it work???
* Wash the column 10ml with the same buffer in two portions (the first run with 5ml, the second run with 5ml).
* Eluete the protein with 10ml buffer (pH=8) with 0.2M NaCl.
* Continue to eluete the protein with 10ml buffer (pH=8.5) with 0.2M NaCl.
* Lastly, eluete the protein with 10ml buffer (pH=9).
or Shouldn't I use NaCl?
or Should I use only pH increase step by step?

Should I use a buffer like that 5mM Na2HPO4, 1mM EDTA (pH=7.5 or 8) ?
or should I use a buffer like that 20mM Tris-HCl, 5mM Na2HPO4, 1mM EDTA?which type of buffer should be used?
AND while I increase the pH, should I add NaOH into the starting buffer in order to obtain a higher pH?

at what pH will you equilibrate the spin column?

since you have found a cation exchange column why don't you try elution with salt instead of pH?