help with his tagged protein purification

[pgrover]

Hi everyone,

I work with two proteins that have a pI of 7.8(A) and 7.1(B) respectively, they are both ~26kDa each and are his-tagged.

I started purifying protein A (pI 7.8) using the GE healthcare hitrap column, charged with Ni ions. I use the following buffers:
Binding buffer: 20mM tris, 0.5M NaCl, 20mM imidazole, 5mM BME, 10% glycerol, pH 8.3
Wash buffer: 20mM tris, 20mM imidazole, 5mM BME, 10% glycerol, pH 8.3
Elution buffer: 20mM tris, 1M imidazole, 5mM BME, 10% glycerol, pH 8.3
I got nice and clean bands from the his column, and then put it on GF column to dilute imidazole out and further purify the protein. I used the following buffer:20mM tris, 100mM NaCl, 3mM DTT, pH 8.3
I got a very broad peak from that and the protein was eluted in ~200mL buffer. I also lost a large amount of protein at this step.
So, next I decided to dialyze the protein into dialysis buffer (20mM tris, 100mM NaCl, 3mM DTT, pH 8.3). Almost all the protein precipitated out of solution (more than 90%).For dialysis, I did 1hr, o/n, 1 hr at 4C.
After I purify this protein, I need to label it with a fluorophore. The optimum conditions for this are pH 6.5-7.5 in a phosphate based buffer.

I started purifying protein B (pI 7.1) using the GE healthcare hitrap column, charged with Ni ions. I used the same buffers as described above.
Again, I got nice and clean bands from the his column, and then dialyzed it under different conditions.
a. 20mM tris, 100mM NaCl, 3mM DTT, pH 8.3
b. 20mM tris, 200mM NaCl, 3mM DTT, pH 8.3
c. 20mM tris, 500mM NaCl, 3mM DTT, pH 8.3
d. 0.1M phosphate, 100mM NaCl, 3mM DTT pH 6.5
e. 0.1M phosphate, 200mM NaCl, 3mM DTT pH 6.5
f. 0.1M phosphate, 500mM NaCl, 3mM DTT pH 6.5
For tris based buffers, I did not get any precipitation.
For phosphate based buffers, I again got a lot of precipitation and lost ~90% protein.

So, I think that the proteins are precipitating because of the pH range. I am in a difficult situation because all pH values are in the same range.
When I move from pH 8.3 to 6.5, the proteins precipitate at their pIs (7.1 and 7.8) But I need the final pH in the range of 6.5-7.5
So, now I have decided to use a phosphate based buffer for the his purification at pH 6.5. When I searched literature, I found that the optimum pH for these columns is 7-8. Some people suggest using low pH for eluting the protein (instead of imidazole). Do you think I'll be fine if I change the pH for all buffers to 6.5 (phosphate based) and continue everything else the same way?
Also, do you have any other suggestions or comments regarding this?

Please reply at the earliest, thanks for your help!

[eeuzc]

Check the Hitrap column manual for the pH that is used for elution. Generally this is very low for Ni columns (about 4-5), so I think your protein would bind to the column at pH 6.5

[fit2page]

you make the right posting

[Kneeanderthal]

I agree with the first post. Try changing the pH of your buffers to 6.5 so you don't have to go through the pI of your protein. You can also try increasing the NaCl concentration to 500 mM when your diluting the imidazole, then dialyze into 100 mM.