I didnt experience as yours, but precipitation of protein during concentration happens with some protein due to various reason. But as you say, there is no aggregation, and it reaches to 3mg/ml and then 1mg/ml?? how do you measure your protein by the way? because I am sure there is no magic and proteins r not volatile :)

If you think it is hugging the membrane, then try with any other filer centrifuges. Some additional details about your protein may really helpful to the people.

As Ananda said, I'd try another filter type/brand. I had a similar issue of a seemingly disappearing protein (it's predicted to be intrinsically unfolded), and it seems that the problem was that the protein was binding to the filter and not coming off. I switched brands and voila! No more vanishing protein (well, much less...went from a recovery rate near 20% to over 70%).

We have experienced similar problems and just tried different membranes and used larger amounts of proteins to start with. In our hands, AMICON ultrafiltration cells work better than miniconcentrators (Ultrafree or similar eppendorf size) from the same brand.

Some times it can also help to just resuspend your protein from the filter. After you have collected the flowthrough, just take a littele bit of buffer on the filter and if its starts foaming you know that you are getting your protein back into solution.

You could try changing the filter material, as the others have suggested. Using some glycerol (typically 10%) in your protein buffer can also help. Another good idea is to stop the centrifuge every 10 minutes or so, and mix the protein sample in the concentrator by pipeting up and down. This would prevent, or at least minimise the concentration gradient in the sample which some proteins don't like.

I had the some problem with protein extracted from liver,proteins was absorbed by membrane during UF.I reduced surface area of membrane and yeld was accettable.

If the alcohol concentration is lower than 50% when you use it to wash the DNA, , DNA will go to the the water and disappear.

Just because you can't see the protein percepitate doesn't mean that its not aggregating. Lots of proteins form high weight soluble aggregates with different spectral properties then free protein. You can check for prescence of soluble aggregates using dynamic light scattering.

Many thanks for the suggestions. The best thing to do is to screen thorough different types of membranes now. We will see!

Regards.

As a few people have already said, there's a good chance your protein is binding the membrane. I work with His-tagged membrane proteins and had a problem similar to this. (I use Millipore concentrators). After concentration I rinsed the membrane in the concentrator with 1 ml buffer and ran it on an SDS-PAGE, which revealed a nice thick protein band. I'd suggest try that rinsing the membrane, and also as Christopher suggested above, use DLS as to double check for aggregation. Good luck!

It is common problem, and i would say it is not binding - it is creation of layer on the top of membrane therefore it is easy to wash out after centrifugation. The standard procedure in this case is to use TF/UF or concentrator with constant mixing and in both cases the technique allows to decrease the layer, but in your case it might not work because you have very small volume to do it.

The PES membrane was developed to decrease protein binding therefore it will be difficult to find another one but you can try.

Agreed with the above thought about agregation issue, it is the most common problem especially during refolding step when part of protein creates dimers and the dimer is soluble, and there are some techniques which decrease agragation and one of them is to add urea or detergent during purification steps but eventually you have to remove them but practically it gives much higher yield.

Your problem is not so easy to solve therefore the only way is try and fail.

I am agree with my colleagues. the protein is surely bound to the ultrafiltration membrane. It happens many times, but as well as to change the type of ultrafiltration membrane you could try with another system to concentrate your protein. You can use the Speed Vac system, do you know? It concentrates under vaccum at low temperature but I suggest this method only if your protein is in a buffer with low ionic strenght and in the case you have no large volumes.

I agree with a number of the above suggestions. I also take aliquots during concentration and measure by DLS. Your protein may be aggregating after a certain concentration. Are you concentrating your protein right away or is it stored at 4C for some time-how stable is your protein?

Do you have a binding partner that could stablize the protein of interest, if so you may want to consider making a complex and then concentrating the sample for crystallization attempts.

Good luck!!

How much salt do you have present in your protein? We typically add 300 mM salt to the final elution and concentrate. Never used the Vivaspin but use the Millipore exclusively so I can't comment on that. The other thing you may consider is ading a low concentration (10%) glycerol.

True that sometimes proteins does bind to the membrane..membrane made of PES is widely considered compatible with most proteins, i guess since you are using a viva spin it means means you want a minimum volume say ~5-10 ml ( if you include the wash it might be even more). like Alessaandra mentioned i suggest you use a Vac system (lyophilizer) if you volume is low.

also try adding glycerol 5-10% to the sample but higher concentration might interfere with your TP estimation.

good luck!

Try exploring different buffer conditions that reduce protein aggregation or membrane binding. I worked with a protein which bound to membranes at 0.3 M NaCl but not 0.5 M NaCl.

try to block/saturate the membrane with certain other proteins like BSA. but make sure it is not interfering with your purity. but worth giving a try!!!!

try equilibratiing you vivaspin column in the protein buffer for several hours before concentrating, I use vivaspin with his-tagged membrane protein and that's what I do, the detergent is binding to the membrane of the vivaspin and significantly decreased the amount of protein that is binded

I'm having the same problem... Protein completely disappeared after a certain concentration (something above 5mg/ml). Not detectable in the flowthrough or the filtrate. Looks like it completely fell out (white clouds after first attempt to resuspend filtermembrane - Amicon flters). At the moment I'm trying to resolve it from the membrane on a spinning wheel with more salt, glycerol and DTT... but I don't think it will still be usable for crystallization experiments after that.

However I want to do the purification again and I was wondering what method I should use instead for concentrating the protein. SpeedVac might be a solution but I have no possibility to use it at 4°C. I would go for dialysis but I'm not sure if I would face the same problems with the dialysis tube as with the Amicon filters...

Any ideas or suggestions?

@Stefan, I'd suggest trying a different filtration device; for my protein of interest, switching from Pierce to Millipore filters (supposedly equivalent products) did the trick. So if you can get your hands on a different device, I'd recommend giving that a try.

I'm not familiar with crystallization, but is acetone precipitation (or other alcohol precipitations) an option?

One more common reason might be degradation of the target protein by proteases and usually protease inhibitor is good enough to prevent it

I think acetone precipitation is not suitable because the protein should still be active and in its "original" state for crystallisation... maybe a salt or PEG precipitation could work... but I've never tried it before - anyone experience with that?

I also got filtration devices from Sartorius... worked before but never tried concentrations above 5mg/ml - and I have to go up to 10mg/ml. If there is a good alternative to membranefiltration I would rather go for that.

I'm pretty sure that I don't have problems with proteases - but at the last step of purification it is not advisable to use inhibitors because the sample shouldn't contain anything more than salts, buffer and protein...

So has anyone experience with concentrating high protein amounts without membranefilters? I would really appreciate any hints!

To my experience your problem is often linked to protein aggregation. Maybe you can try to use Amicon stirred cells from Millipore. Instead of centrifugal force, the stirred cells work with high pressure gas plow. You may observe any of sample changes during the concentration process. With such observation you might be able to nail down the problem as well. Inhibitors or binding partners often can be used to help to prevent aggregation. After solving the aggregation problem we did use these devices to bring proteins to very high concentration.

Thank you for your answer. Never heard of the stirred cells before - very interesting. In the mean time I tried dialysis against PEG 6000 and it worked fine in the first try. I will repeat this one more time before I use it in the main experiment - but looks promising. I increased the concentration from ~3mg/ml to 12 mg/ml within an hour (350µl volume to 83µl). That was with a loss of a few % of the protein but thats ok I guess...

• Hai Stefan, Dialysis again PEG 6000? Can you please explain to me the procedure. I'm having the same problem as you