I think at pH 4, since the PI is 1.8 where it is neutral, the carboxyl groups become protonated so your protein becomes more positively charged. Therefore it would not likely stick to a Q column which is made of quaternary positively charged amines. You would need a negatively charged column like CM cellulose, MonoS, etc. Just look up cation exchange columns.

Sorry Marcia, but you are wrong : the protein is negatively charged if the pH of the buffer is over the pI. So normally, it should bind to the Q column.

Tatiane, do you see any aggregation of your protein before applying it to your column ? Is there a particular reason why you use a buffer at pH4 ? Because if your protein isn't known to be active only at low pH but rather at neutral pH, then using pH4 as a buffer will protonate lots of chemical groups in your protein, which can unfold your protein and make it aggregate.

Second, your loading buffer doesn't contain any salt, which normally should increase the binding to the ion exchange column, but can also make your protein aggregate. Remember lots of proteins don't like to be in buffers devoid of salts, even if the presence of salts can of course lower or prevent the binding to your ion exchange matrix.

I agree with Yann. Is it a DNA binding protein? Do you see a peak in your elution profile at 350-400mM salt? Because DNA binding can also promote aggregation of your protein. If its the case you can get rid of DNA by adding DNase in your lysis buffer or you also can do a negative Q column where all the DNA is binding to the Q column and you collect the flowthrough.

I would try a range of more physiological pH as your PI is really low anyway, it should not hurt the binding to the Q column. And I would put salt in your dilution buffer something like 70mM. As Yann said, proteins do not like acidic pH and buffers without salt usually.

Good luck

Hi all! Thank you for the replies. Yann: my protein is known to be stable in pHs between 4.0 and 8.0. It is a serum protein and I tried to work at a low pH in order to precipitate the other proteins present in the preparation, so that I have preferentially my protein binding to the matrix. In fact, my loading buffer is the pteparation diluted in buffer A, so it do has salt in it.

I did what Nicolas suggested and used less salt in loading buffer (I diluted ten times my Buffer A, so samples were loaded in NaAc 10mM instead) and got a peak, but it is still a large one. Today I'll try to use pH 6.0 to see if I augment the binding and recovery.

Thank you all very much.

I'll come back latter to let you know how things are going on hplc.

Take care,

Tati.

Yes you are right. I am wrong. At pH 4, the carboxyl groups will become deprotonated and the protein will become less positively charged.