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a few questions about ion exchange chromatography


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#1 xaxax

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Posted 02 November 2011 - 01:53 AM

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.

Edited by xaxax, 02 November 2011 - 01:55 AM.


#2 allynspear

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Posted 02 November 2011 - 06:47 AM

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.

#3 K.B.

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Posted 02 November 2011 - 01:02 PM

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.

Correct.

* 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?

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

OR if use a cation exchanger, the buffer pH=8 is enough?

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

*** 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???

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.

* It is known that increase in salt gradient releases the bounded proteins from ion exchange resins. why NaCl is used?

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

Should it be dialysed? what should the dialysis buffer content be?

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.

** What is the aim of these chemicals used in these steps?

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

#4 xaxax

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Posted 02 November 2011 - 01:11 PM

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.

Edited by xaxax, 02 November 2011 - 01:50 PM.


#5 xaxax

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Posted 03 November 2011 - 02:19 PM

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...

#6 mdfenko

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Posted 04 November 2011 - 06:46 AM

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.

Attached Files


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#7 xaxax

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Posted 06 November 2011 - 03:53 PM

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?

#8 mdfenko

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Posted 07 November 2011 - 07:58 AM

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.
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#9 xaxax

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Posted 10 November 2011 - 07:40 AM

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?

#10 mdfenko

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Posted 10 November 2011 - 07:51 AM

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?
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#11 xaxax

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Posted 12 November 2011 - 11:31 AM

I will equiliprate the column at pH= 7.5 or 8. I think if I use a small quantity of salt with increasing pH, i will help more efficiently. However, you know the technique and have experience more than me. Therefore, I will make what you say.
For a cation exchange, is it more useful to take elute by salt gradient?
And, I want to learn that when I want to make salt gradient, how far the buffer pH should be below the pI.
In all salt gradient applications for taking the desired protein in eluate, buffer pH and content must be constant, mustn't they?

#12 mdfenko

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Posted 14 November 2011 - 09:34 AM

For a cation exchange, is it more useful to take elute by salt gradient?

yes. there is less chance of precipitating the protein in the column.

I want to learn that when I want to make salt gradient, how far the buffer pH should be below the pI.

1 pH unit should be sufficient

In all salt gradient applications for taking the desired protein in eluate, buffer pH and content must be constant, mustn't they?

yes, for most applications. sometime chromatography techniques may improve if more than one gradient runs concurrently (eg- hydrophobic interaction chromatography elutions sometimes require increasing ethylene glycol while decreasing salt).
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#13 xaxax

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Posted 23 November 2011 - 05:52 AM

MDFENKO, I want to thank you again. Your help was very crucial for obtaining the desired protein.
I want to write the procedure what I did and what I eluted.
Starting Buffer: 20mM Na2HPO4, 1mM EDTA (10ml, pH=7.40) pH was arranged with HCl or NaOH.
Wash Buffer: 20mM Na2HPO4, 1mM EDTA (10ml, pH=7.40)
Elution Buffer: 5mM Na2HPO4, 1mM EDTA, 100mM NaCl. pH of elution buffers were adjusted to 8, 8.5, 9 and 9.5, respectively, by means of NaOH or HCl.
5ml starting buffer was loaded to the column and centrifuged at 500g for 5 minutes. pH of the washing buffer changed to near 4.5
Again, 5ml starting buffer was loaded to the column and centrifuged at 500g for 5 minutes. pH was 7.40

Then, one quarter of protein solution (4ml) was loaded to the column and centifuged at 500g for 5 minutes. The remaining protein solution was loaded identically and all flowthrough was collected and combined. Then, combined flowthrough was loaded to the column, again. Then, flowthrough was taken and pH of this was 7.25.

The column was washed twice (2x5ml) with Wash Buffer. pH of the wash buffer taken from the bottom of the column was 7.40.

To elute the desired protein, half of the first elution buffer (pH=8, 5ml) was applied to the column and centrifuged as mentioned above. After first load, the remaining half of the first elution buffer was applied and centrifuged. pH of the first run was 7.28 and the second one was 7.60.

This elution procedure was carried out with the second (pH=8.5), the third (pH=9) and the fourth (pH=9.5) elution buffers and the obtained pH values were as written below respectively:
7.82 7.86 7.84 8.14 8.08 8.36
When the eluats, flowthrough and wash applied on SDS PAGE, I saw the desired protein in the first and the second eluate. Also, a small amount of protein was observed in flowthrough and wash. No protein was observed in the third and fourth eluate.
However, there were 5 or 6 visible protein lines that were observed on SDS polyacrylamied gel. Maybe I use an anion exchange or GST resin to obtain the desired protein.
Can I use my column again? Does it work?

Edited by xaxax, 23 November 2011 - 05:55 AM.


#14 mdfenko

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Posted 23 November 2011 - 08:51 AM

switching to an anion exchanger will not make much of a difference. why do you think gst will work for you (and if it would then why didn't you use it in the first place)?

it is extremely rare (if at all) for all of the protein to bind. so, seeing some of your protein in the flow through and wash is nothing to worry too much about (you may have exceeded the binding capacity of the exchanger, some of the protein may be denatured, random effects, etc), as long as the bulk of your protein bound.

you should ensure that your washes and elutions are complete. final wash should contain no protein. final elution should be at the proper pH and contain no protein.

why did you reduce the concentration of buffer in your elution solutions? they won't attain or hold pH as well. also, ensure that the buffering agent you use is effective in the pH range you need (you may have to blend buffers to maintain buffering capacity). rule of thumb, buffering agents buffer at + or -1 pH unit from the pK. phosphate has 3 pKs but they don't overlap.

if your protein elutes pure (or nearly pure) and reasonably completely then you are successful and don't need to try another matrix (you could do some final polishing with gel filtration if you have a few contaminating proteins present and they're enough different in molecular weight).
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#15 xaxax

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Posted 24 November 2011 - 05:23 AM

excuse me, I wrote the pH of the buffer wrongly. It must be 50mM Na2HPO4. I increased the pH to ensure a stable pH.
Also, after I dried the SDS page, I saw 9 or 10 protein bands Posted Image (At first, I had seen 5-6 lines before I dried the SDS).
In literature, I saw GST is a good way to obtain the desired protein, but there must be a few proteins. Now, I don't know what I sould do.
Thanks for your all friendly suggestions.

Edited by xaxax, 24 November 2011 - 05:26 AM.





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