Hi have you tried arginine as an additive. We find that if it is interactions with the column addition of 0.2-0.5M arginine sharpens the peaks up. There are several papers in the literature on this property of arginine.

Thank you Jonathan, you're totally right about the proteins eluting later, I had seen this before and I'm not really concerned about this apparently lower MW. Actually in the first purification I used 0.15M NaCl and since this retention occurred I used 0.3M NaCl in the second purification but the same thing happened. Maybe it is due to the interaction with the resin, true. But what surprises me more is the fact that it elutes in so many fractions (even at volumes corresponding to much higher MW). In other cases, even when the amount of protein injected into the column was massive I used to get wider peaks at the base and the total volume of the fractions containing the protein was around 20ml max. With this domain I have even 12x5ml fractions, all of them with similar amount of protein (except those corresponding to the 30kDa peak which contain more protein)...

The pumping of the AKTA should be OK, the fraction volume is 5ml and I don't detect any air in the systems.

Hello Marcin,

I have few words as suggestion toward wide range of elution volumes.

Even you know your protein forms complex and you dont suspect it to form diffferent oligomerization states. But, in some case if you concentrate more than the limit in the given condition, your protein start to form different oligomers. I faced similar problems. when i concentrated more prior to gel filtration, it forms different oligomerization and i used to get very broad not very symmetrical peak. And, when I checked my protein quality by Dynamic light scattering technique I found my protein sample highly polydispersed and in this study i also found the concentration limit. After when I did gelfiltration again with my protein sample within concn limit i got one nice symmetrical peak. So, you can check whether your protein quality is really good or going worse upon concentration. You can obviously play with different buffer condition where your protein is more stable and less polydispersed.

Thanks Shiv. Definitely DLS would be a nice thing to try with this protein but I don't have access to this technique in my institute. I've been doing some additional trials with different buffers and pHs and I think that this broad range of elution might be caused by different oligomers and unspecific interactions with the column resin. I managed to get one quite nice peak at a volume that corresponds to the MW of the monomer at pH 8 even though the theoretical pI of the protein is around 8.5 but somehow it behaves better at 8 than at lower pH (6-7.5) at which it used to give me a peak corresponding to a smaller MW with an asymmetrical shoulder towards higher molecular weights. I don't clearly understand this behavior... Another interesting thing I observed is that when I checked the complex between this domain and its binding partner which never gave problems when run alone in SEC when bound together the elution profile was weird again and both proteins co-eluted in many fractions in a broad range of volume. Crazy! Anyway, thank you for the useful comments!

Hi have you tried arginine as an additive. We find that if it is interactions with the column addition of 0.2-0.5M arginine sharpens the peaks up. There are several papers in the literature on this property of arginine.

Hi John! That's an interesting suggestion. I was aware of the role of arginine in preventing protein aggregation but didn't know it could also help in the case of column-protein interactions. I'll have a look on that!

Hi Jonathan! I have been experiencing similar issues as yours. My protein of interest is 13KD and its peak comes at 140 ml in my Sephacryl 16/60 column (120ml volume). I am curious if you have ever tested whether this affects the purity of the protein?

Do your other proteins elute at the volume as expected? All of my peaks under come out later as expected. I purify my protein from whole cell lysate of E.coli. Do you think other components in the lysate such as lipids or DNA might contribute to this issue?

The interesting thing is, when I add BSA to the lysate, the BSA peak comes out at the expected volume (~50ml), so it seems that only proteins from E.coli lysate are experiencing delaying problem.