31 replies to this topic

[mdfenko]

you could try gel filtration to polish your protein.

[xaxax]

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?

Edited by xaxax, 29 November 2011 - 01:31 AM.

[mdfenko]

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.

[xaxax]

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?

Edited by xaxax, 29 November 2011 - 10:24 PM.

[mdfenko]

Do you suggest me to add buffering salt like Na2HPO4 or NaH2PO4? OR both of them?

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.

[xaxax]

I want to do your suggestion about equilibration with dialyzing, but can I use my pre-used column again. It is Sartorius Vivapure Maxi S column (CATION COLUMN).
How can I regenerate my column?
I found 3 methods. Which of them is convenient for my column? and for regenerating columns?

1) wash column with 4 bed volumes 0.5-1M NaOH, then with 2-3 bed volumes pf water
wash with 4 bed volumes of 70% ethanol followed by 4 bed volumes of water
wash with 2 bed volumes of 0.5-1M HCl and 4 bed volumes of water
wash with starting buffer to equilibrate

2) wash column with 70% ethanol then with water
wash with 0.5-1M HCl and with water (to neutralize)
wash with 0.5-1M NaOH and with water (to neutralize)
wash with starting buffer to equilibrate

3) wash with 70%ethanol
if it is a cation column,wash with 0.5-1M HCl and water
wash with starting buffer to equilibrate
or if it is an anion column, wash with 0.5-1M NaOH and water
wash with starting buffer to equilibrate

[mdfenko]

i just wash with high salt and re-equilibrate between uses.

i only regenerate when capacity significantly diminishes. then i regenerate by cycling with acid and base (flushing with water in between), the order depending on whether it is anion or cation exchange.

never saw procedure using ethanol.

there is a method to clean the exchanger given in the handbook.

[xaxax]

the handbook suggests to wash with ethanol. It says '' when it is stored after using, it should be in ethanol''.
since i use cation column, firstly, i should wash with acid (to remove the unwanted bounded proteins) and water (to neutralize). After that, I should wash with base and water. Then, I should equilibrate the column with starting buffer. ???

[mdfenko]

as you say, the ethanol is for storage (and should be 10 or 20%), not to wash or regenerate.

the scheme you laid out to regenerate looks fine, just make sure that you don't exceed the recommended concentration of acid/base for your matrix. make sure you flush with a large volume of water between the acid and base treatments.

as i said before, high salt washes and re-equilibration are sufficient most of the time (until binding capacity has significantly diminished).

[xaxax]

Hello Mdfenko
I want to ask something, again. :-)
This time, i want to make gst purification, but I want to learn 3-4 important and basic question by means of your knowledge and experience.
1) What should the pH of protein solution to be loaded be?
2) What temperature should be used for gst purification? It is suggested both 4 C and room temperature. According to the temperature, the pH of equilibration and elution buffers will change.
3) Are there any elution buffers except for tris buffer?
4) Some articles suggest PBS for equilibration and washing the resin. And some suggests tris buffer for equilibration, wash and elution (with reduced glutathione).

[mdfenko]

1) a pH where your protein is stable and in solution (around neutral, if possible).

2) i always prefer to run at 4C but if run rapidly (e.g. if run on hplc or fplc) and the fractions are put on ice immediately then room temperature should be okay.

3) depends on the pH at which you run the column and the buffering range of the buffer. as you point out in your next question, phosphate buffers work fine.

4) use whichever suits your needs better.

you may want to download these 2 handbooks from gehealthcare life sciences:

gst gene fusion system

and

affinity chromatography

[xaxax]

Thanks for your feedback. I applied the experiment at room temperature. I used PBS buffer to equilibrate-bind-wash steps. My elution buffer was 50 mM Tris-HCl containing 10mM glutathione.
Wash was made with 10 bed volumes twice. Elution was made with 2 bed volumes three times.
SDS page image was like that:
There was no loss of the desired protein with Flowthrough and Wash steps. However, in the elution step, the desired protein could not be seen. I thought that the protein might have been in the resin, then, I applied the elution buffer containing 20mM glutathione, however, I could not see the desired protein.

[mdfenko]

what is the salt content of the elution buffer?

is there more salt in the equilibration buffer than in the elution buffer?

routinely in ion exchange chromatography, elution buffers are equilibration buffer with higher salt concentrations. you may want to add salt to your elution buffer so that it is more concentrated than the equilibration buffer.

[xaxax]

Hello MdFenko
My equilibration-wash buffer contains 4.3mM Na2HPO4, 1.47mM KH2PO4, 137mM NaCl, 2.7mM KCl.
and the elution buffer contains only 50mM Tris and 10mM glutathione (pH=8)
These buffers are suggested by the GST resin supplier.
I equilibrated the pH of the equilibration-wash buffer to the pH of the protein solution.
After I carried out ion exchange purification, I want to polish my protein with gst purification.
In my opinion, the equilibration-wash buffer and the elution content for the GST purification should be the same as the protein solution which is eluted from ion exchange. Content of the protein solution-taken from ion exchange- is 100mM NaCl, 5mM Na2HPO4, 1mM EDTA.
In addition to this, I think that the working pH should be same with the protein solution-taken from ion exchange- in every step of GST purification.

Edited by xaxax, 10 January 2012 - 01:45 AM.

[mdfenko]

you will still want to add some salt to the elution buffer to prevent non-specific binding to the matrix.

polishing is normally performed by gel filtration. affinity is a further purification step (and probably should have been done first).