Hello Luiza

I would try a small ultrafiltration cartridge,, e.g. Vivaspin 500 and pass your prep in several batches. Keeping the volume of the cartridge as small as possible should minimize losses due to adsorption to the walls and to the ultrafiltration membrane.

I see. What do you mean keeping the volume to as little as possible? As in letting as much filtrate to go through as possible? The vivaspin has a 5ul deadstop, should I spin till I reach it? Why this would minimize loss due adsorption? Wouldn't this make things worse? I'm sorry if those are stupid questions, I just want to have as much info as I can, the protein is really valuable!

Would you recommend a 10kDa, 5kDa or 3kDa cut-off?

Hi Luiza,

If the protein is more than 95% pure, I will prefer 3Kda cut off spin tube to retains as much as protein, that too after passing the buffer couple of time to avoid protein adsorption on the membrane. Personal experience says there is always a loss of protein, no matter whatever best available resource you are using.

Best

Using the ultrafiltration device would also be my first choice, unless you have detergent in your protein sample at a concentration above its micellar concentration. In that case, concentrating the sample will also concentrate the detergent. The molecular weight cutoff should be smaller than the molecular weight of the protein. Lower cutoff filters take longer.

Another approach you could take is lyophilization (freeze-drying). However, this method should not be used if the protein is unstable to drying, or if concentrating the solutes is a problem.

I would go with ultrafiltration as well. As per Pierre's suggestion, use as small a size filtration device as possible. In your example, you have 4ml and need to get to 1ml, then using an Amicon ultra 4 would be fastest, but you could use a filtration device with smaller total volume such as the ultra 0.5 and proceed in batches, keeping the filtrate in the device as you add more dilute protein solution. A 10kDa MWCO should be sufficient to retain your protein. If you protein is well-behaved, your losses should be less than 5% with this system. If it were me, I would go with the 4ml unit.

It is sometimes helpful to repipet your material several times when recovering it from the device. You could also concentrate to 500ul then rinse the device with 500ul buffer to ensure maximum recovery for your final volume of 1ml.

I would like to add that using affinity columns to concentrate proteins is a good choice if you have large volumes of dilute proteins, for example, eukaryotic expression supernatant, and you need a high factor of concentration as well as purification. If your protein is already in a small volume and relatively pure and you need only a small increase in concentration, this is probably not the way to go, given that you have to elute in a buffer that contains solutes which needs to be removed, in the case of Ni/NTA, Imidazole, for example, meaning you need a buffer exchange step as well, either SEC column, buffer exchange column or dialysis. Thus you need 2 steps at least. The reason I suggested ultrafiltration is that it is only one step and you are there.

I would be interested to hear what you did and how well it worked.

Thank you everyone for the suggestions. I found out we already have some 3kDa Amicons here in the lab so I'll probably use those using the tips described here, concentrating in batches, repipetting multiple times after each batch and using a small amount of buffer to rinse the column. I'll let you know how it went afterwards! Thanks!