Hello guys, I have recently purified a his-tagged protein via Ni beads. Based on a BSA standard, it was estimated that the eluted protein concentration was about 1 microgram per microlitre in 100 microlitres of PBS pH 8.5. However, upon dialysis (to remove imidazole and exchange buffer to Hepes pH8.5), the eventual protein concentration became 0.1 microgram per microlitre in the same final volume. The buffer exchange was performed as the purified protein will be subjected to a phosphate dependent assay and hence the need to remove PBS. However, I do not understand what might have caused the 10 fold reduction in total protein amount. The pH was carefully controlled to be away from the PI of the protein and even EDTA was added during elution to prevent potential aggregation that may occur during Ni leeching. Also, protease inhibitors were added from the start till elution and I suspect proteolysis is unlikely too. I am considering using Hepes from the start but do offer some comments on what I might have missed out. Thanks.
How big is your protein? A common mistake is to take the filter's stated cutoff at face value. It's a rather broad blurry line, not a sharp cutoff. I often see improved recovery if my protein is as high as 3x the cutoff.
If your protein is aggregated and not in soluble form, it may get stick to the membrane in the Amicon filter. I will recommend you to use a desalting column for buffer exchange.
Hi John, my protein has a size of about 25kDa, which is slightly more than twice the cutoff size. Boopathi, I performed Amicon filter to concentrate the protein. The exchange was performed via dialysis when I used Hepes buffer in the reservoir. Though I saw no precipitation, there remains a possibility that they might have been too small to be observed. Indeed, there's the likelihood that they are trapped in the Amicon filter or even in the dialysis bag. Thanks.
Hello, I agree with Boopathi that desalting columns are the best choice for buffer exchange (fast and highly efficient). As for protein concentration we faced the same problem, our protein was lost on Amicon filters, most probably attached to the filter. However, the problem was overcome when we changed Amicon filters for Vivaspin concentrators, which are more expensive but work fine
Have you tried to leave out the dialysis part and to change the buffer with the Amicon? Each step is reducing your yield, more or less. But probably your down-stream assay is too sensitive against remaining phosphate, right?
Hi Marina, I will definitely give the desalting column and vivaspin a try. Vivica, I have heard of that approach before but the trace amount of phosphate is indeed a concern for my experiment. Thanks.
Hi Adam,
It's a fact of life that you lose protein at every single step of purification. The worst culprits seem to be gel filtration and concentration, though gel filtration isn't as bad as people think if you take into account the loss includes all the contaminating proteins (which were certainly included in the total protein concentration calculated before loading the column).
Anyway, I digress. Back to concentrators: stay away from any concentrator that uses regenerated cellulose as these are notorious for binding to many proteins. Try concentrators that have PES (polyethersulfone) membranes as they significantly reduce this non-specific binding problem. They are slightly more expensive but definitely worth the extra cost. We use Vivaspin concentrators, just make sure you choose the ones that have PES membrane as they produce concentrators with several membrane types.
Another type of membrane that has been discussed in other post is nylon. According to many this is even better than PES. I have not searched extensively for these yet, so if anybody knows of a good product that uses nylon membranes, then please let me know.
Hey Antonio, 'fact of life' is definitely a good way of putting it. I will go check out on the various membranes as I never knew that there can be so much complications behind the type that is used in concentrators. Thank you for bringing these up.
Are you sure your buffer's pH is proper? Hepes' useful pH range is 6.8 - 8.2 with pKa 7.56... For phosphate is also too high. For 8.5 I would use Tris-HCl. It is possible that your buffer's pH is unstable and it's the reason why you are loosing your protein. Haven't you noticed precipitation?
Best Regards!
Hey Dawid, I agree with you that I might have pushed the limits of the 2 buffers that I used. Though I saw no precipitation, it is possible that it might have been too small to be observed. TBS is something I would try as the both the buffering range and the absence of phosphate might make it the optimal buffer for my experiment. Thanks man!
- Badges
- Science method