Hello,
I'm using a Q Column for ion exchange chromatography protein purification. It's becoming a recent problem that when I try to elute my protein using a NaCl gradient my conductivity increases sharply at the beginning of the chromatography (after 4mL). At the same time, a UV signal appears simetrycally along this conductivity peak (figure). Thus, proteins aren't separated and all elute within this fraction.
It is very unusual since it is not the first time I purify this protein and this is a new column. Any thoughts?
thank you!
José
Hi there,
Your protein sample is far too concentrated in salt which hampers most of the protein matrix interactions. So what you should do is to dilute your protein sample with low concentration buffer (20mM Tris for instance) until salt concentration (NaCl most of the time) is less than 50mM. Column should be equilibrated with this low ionic strength buffer. After sample loading this low ionic strength should be maintained until UV signal comes back to the baseline. And then salt gradient should be applied.
What is the salt concentration in your sample? Including buffers
Difficult to tell without seeing the whole logbook, but an educated guess is that for some reason you have some elution buffer in the mixer chamber when the elution starts. One reason could be that you are doing a pump wash? If so you should do the pump B wash first followed by pump A. Another reason could be a faulty flow restrictor in the B pump. There is a spring loaded membrane located in the black manifold between the two pumps. It is there to provide some back pressure to ensure good seating of the back valves and to prevent buffers from running by themselves through the system. If that flow restrictor has stuck in the open position your B buffer can run freely. An effect will be that during sample loading using the sample pump buffer B will slowly run into the flow path and when you the start washing/elution you will have buffer B in the mixer chamber. We have had that happen on a few systems. In one case there was a long (over night) sample loading and during this time all the buffer B drained out. And the column was eluted with air!
Lower salt concentration in final buffer (B). Good wash column after application probe (3-4 vol. of column or more).
Not enough information to give you a good answer. Tell us all the buffers you used (Sample, A and B), the Vo and Vt of this column and from the picture I can't tell if the protein peak is big or small, what's the peaks value in mAU? Are you measuring at 280nm?
Bo Pontoppidan had very good suggestions - Below I fill in his response with more detail.
Hernán Bucci I agree with as well - Below I'm shooting from the hip with a quick answer which may miss the target.
You check the conductivity of the sample prior to chromatography - rule of thumb is to have the protein sample at the same conductivity as the lowest point on your elution gradient. Usually, the starting point is to dialyze or desalt 10 mM PB. I apologize if you are already doing this - I just didn't have that information from your description.
Best Regards
Dan
Hi! Thank you all for your answers, I did check each of them. I know it's been a while since this problem but I'd like to post the answer for the record.
The problem: a sharp increase in conductivity at the begining of the elution process. This should not happen since it is a gradient eltuion, thus conductivity must increase gradually to obtain adequate protein separation.
Solution: Following Dan suggestion, I concluded that the buffer used for protein extraction was causing the conductivity problems. This buffer contains EDTA, which if not prepared appropriately can cause variations in conductivity and moreover can bind the ion-exchange column. The problem was solved when I prepared my extraction buffer using a stable stock solution of EDTA.
Sorry for the awfully late reply, but this might be useful information if you encounter similar problems.
Thank you,
Jose.
Jose, what method of preparation of the EDTA stock solution did you find to be appropriate?
I think it depends on the buffer concentration in the first suggestion and the second the problem may be in the column wich should be washed precisely from the west of past elution
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