Chromatography Forum

Yesterday I'd try to purify a pyridoxal enzyme namely Glutamate decarboxylase (GAD) which was extracted from Aspergillus oryzae by using weak anion exchange chromatography column (Hitrap capto DEAE) , 1ml column that purchased from GE healthcare. Following are the running condition and buffer I'd use:

Starting buffer: 20mM L-histidine buffer (As suggested from the column's manual), pH5.5
Binding buffer: 20mM L-histidine buffer + 1mM NaCl, pH5.5
Flowrate: 1.0ml/min

Unfortunately, almost 95% of the GAD enzyme was elute out during the equilibration stage and only a relatively low amount of GAD activity was detected during the gradient elution. Therefore I'm seeking for any suggestion which may help me to improve my purification work! Thank you! Maybe some suggestions for the buffer pH, type of buffer, or running condition! Are buffer pH and buffer types play significant roles on this issue?

Try to adjust the buffers' pH to 6.0. If no improvement go to pH 6.5. And finally 7.0.
One of those pH values (or some between) should be adequate.
Remember the sample should be dissolved in the binding buffer at the same pH.

Best regards

Thanks Danko.

Would it make any difference if I select Tris.HCl or Triethanolamine as alternative buffer with the pH range as stated by you?

Not much apart from the fact that TRIS' pKa is ca. 8.3 which means its buffer capacity is within the pH 7.3 – 9.3 range. So, if the initial tests (i.e. pH 6.0 – 6.6 – 7.0) show no or little improvement you might as well go up to pH 8ish and then it'll make sens to use TRIS.
NB. It's important to operate in the buffers' capacity range – not least in ion exchange context.

What is you elution buffer by the way?

Best Regards

I was using L-histidine elution buffer at pH 5.5. Didn't really observe any binding occurred so I was thinking of switching to higher range of pH with TRIS as buffer. I shall update again if it works.

Assuming if I'm planning to test out pH at range between 6 - 7, would triethanolamine be a good choice? Any other buffers can you recommend?

Thanks for your advice, Danko!

You are welcome.
Triethanolamin's pKa is ca. 7.9 (can't remember exactly)? So it's not a good option either.
For the relatively lower pH values you might use sodium phosphate – perfect for the pH 5.9 – 7.9 range. It has to be of low concentration i.e. 0.02 M for instance.
As for the higher pH option (pH TRIS will be perfect.

Your elution buffer contains some sort of salt in a higher concentration – right? - (f. ex. 0.5 M NaCl) in addition to the buffer component. If not, the problem is probably there – you simply don't elute the protein and it stays on the column.

Best Regards

NB. The smiley in my previous post is some sort of a bug. It should be the digit eight.

best Regards