Chromatography Forum

Hi

I need a little help with anion exchange chromatography.. I am trying to purify a protein (pI might be around 5.5) using Q-sepharose column. While trying to optimize the binding pH, the sample was in 50mM phosphate buffer pH 7.5. The protein bound to the column equilibrated with buffer pH 7 and 8, but not with pH 5 and 6. Any suggestions on what pH to use?

Aren’t you just lucky: 50 mM phosphate at pH 7.5 looks like the perfect bid for your equilibrating buffer (I typically recommend a couple of pH units away from pI). Phosphate has its second pKa at 7.2, so you can just hold on to it.
Regarding elution buffer; you can try and add 0.5 M NaCl to the 50 mM phosphate and this would be eluent B. You can also try 50 mM phosphate for eluent A and 0.5 M phosphate for eluent B (both at pH 7.5). But NaCl is typically the best option for elution without any risk of precipitating the protein.

Best Regards

thanks!

btw, i have another query.. how does the ionic strength of the buffer affect protein binding? For instance, I used 50mM phosphate buffer, but how would a 10mM buffer change binding of the protein? And does using phosphate buffer for anion exchange cause any problem.. or should I use Tris (for long term use of column)?

You were welcome.

Regarding the additional question; the binding is inversely proportional to the ionic strength. So, if you reduce the buffer concentration to 10 mM you probably will achieve a stronger biding and higher loading capacity. But remember that the buffer (i.e. pH control) is essential in this context, so you can’t just reduce it to nothing.
Finally you can easily go for TRIS instead of phosphate but in that case I’ll recommend a bit higher pH – for instance 8’ ish.

Best Regards