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

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?

xaxax on Sat Nov 12 19:31:38 2011 said:

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

1 pH unit should be sufficient

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).

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?

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).

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 (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.

you could try gel filtration to polish your protein.

If I obtain good results, I will write them to this board. Maybe this helps someone.
I want to ask you something again.
pH of my protein solution is about 8.20. However, I equilibrate the column with pH=7.4. Should I pour my protein solution to the equilibration buffer in order to obtain the pH of protein solution near 7.4?
What must be done when pH of a protein solution is above or under the pH of equilibration buffer? (I use equilibration buffer when i wash the column after flowthrough) If I do this, can protein precipitate?
For example, I want to use cation exchange chromatography. pI of my protein is 7 and pH of my protein solution is 7.50. What should be done?
OR should I add HCl or NaOH to the protein solution to bring to near the pH of equilibration buffer?

you should adjust the pH of the protein solution to that of the equilibration buffer. you can dialyze so that you don't significantly change the sample volume. i wouldn't add naoh or hcl, too harsh. use concentrated buffer salts instead.

are you using the same protein as before? before you said that the pI is 8.21 now you say 7. if you want to switch to cation exchange then the pH has to be lower than the pI.

I am using the same protein, but as I said the pH of the protein solution is above the equilibration buffer. Do you suggest me to add buffering salt like Na2HPO4 or NaH2PO4? OR both of them?

xaxax on Tue Nov 29 21:21:05 2011 said:

yes. keep in mind that you will be increasing the ionic strength of the solution. this may affect binding to the matrix.

you can dialyze against the equilibration buffer to adjust the ionic strength.