Hello!
I perform protein purification on a weekly basis and I have been really struggling with some proteins in terms of concentrating them. For all proteins I use centrifugal concentrations from the same brand, which work fast for most of them (usually within 2hours from 25mL to 5mL). But for the rest nightmare ones, it can take as long as 8 to 10 hours to concentrate.
I know people recommend trying concentrators with different membrane materials, but there is too much trial-and-error involved and more importantly, it is usually too late when we realise we again use a "wrong" concentrator.
I was instead thinking of doing some small-scale trials to see how protein's solubility improves under different buffer conditions (e.g., salt concentrations, arginine&glutamic acid, ATP), hoping this can solve the prolonged concentration problem. So I wondered if anyone has had any successful experience of increasing protein solubility to work around this insanely long concentration process? Thank you very much!
I assume that you need the obtain the concentrated protein in native condition. Otherwise, you may use urea, guanidine for increasing solubility. In the case of aiming higher solubility at the native form, you may prefer to use non-denaturing detergents at low concentrations. As you indicated salting in approach can be suitable for proteins. Do you observe aggregation during concentration?
It looks like your protein is aggregating and clogging the ultrafiltration device. This most likely has to do with the protein itself and not with the nature of the ultrafiltration membrane. Unfortunately, this is rather common for many proteins. Your best bet is to find by trial and error conditions that improve the solubility of your protein without denaturing it. If possible, stay away from detergents: they form micelles that will not pass through the ultrafiltration membrane and accumulate in the concentrate.
Membrane concentrators often set up a "gel layer" near the membrane surface and the protein collected there does not allow liquid to pass. This can often be visualized as a difference in refractive index near the surface, or coloration. Vortexing intermittently can sometimes greatly increase the processes speed.
For larger volumes another old school trick is putting the dilute protein solution in a dialysis membrane and then dumping dry PEG of a MW at least 2x larger then the membranes MWCO, over the bag while laying in a clean pan (I've used over sized Petri dishes). Come back in an hour or two and the bag has shrunk markedly, rinse the thick PEG goo off the outside and isolate the much more concentrated protein from inside the bag. Good luck!
As the protein concentration increases, the protein will adhere to the membrane of the concentrator, making further concentration difficult to perform. I usually use a pipette tip to gently blow the protein on the membrane to make it evenly distributed in the solution. This is an effective way to shorten the concentration time.
What volume are we talking about? Perhaps, an Amicon cell (https://www.sigmaaldrich.com/technical-documents/articles/biology/amicon-stirred-cells.html) would work better, as the solution is constantly stirred, so the protein will not form a layer on the membrane. However, you'll need a final volume of a few mL.
İsmail Emir Akyildiz Thanks for the suggestions. Yes, we need the protein to be in their native forms. Using detergent is a good idea, but I heard that they do not pass the concentrator membrane? Yes, I will see how adjusting salt concentrations works for my proteins ..
In terms of protein aggregation during concentration, I do have seen flocculate like stuff rising up from the concentrator membrane when I resuspend the solution. Also, I have compared samples right before the concentration step and right afterwards, and it turns out the protein of interest was selectively lost relative to other co-purified proteins in the sample. So I hypothesise that the protein forms aggregate during the process and its aggregate blocks the concentrator membrane.
Pierre Béguin Thank you for the information! Yes I do have heard about detergents accumulating with the protein. My way to assess protein solubility is to compare its levels in the supernant and in the pellet after 20,000g centrifugation. But I understand this "solubility" may not correspond to how much the protein is prone to aggregate when purified? Do you have some suggestions regarding how to more properly test protein solubilities under different conditions? Thanks!
Hi Edward Michelini Thanks a lot! Some people I know of are indeed using dialysis to concentrate their nightmare proteins. But I also understand from them that this approach maximally concentrates proteins by 3-4 folds? Because my proteins have extremely low expression levels to begin with, so I probably need more powerful ways to concentrate it. But thanks!
Engelbert Buxbaum Thanks for the advice! Just had a brief look into the technique and it is new to me. Normally we have several millilitres of solution to concentrate so this should not be a problem. But I guess (just from the animation they provide) I will need a big external pressurisation equipment ...?
No, just a bottle of nitrogen with a needle valve. Because you need only a few bars, and hardly any volume, it can even be a mostly empty bottle.
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