Depending on your sample volume, centrifugal filter concentrators from Millipore, Corning, Pierce etc are a good choice. For larger volumes I prefer to dialyse my sample using Pierce's SnakeSkin tubing followed by lyophilisation (freeze-drying). That way you can resuspend your protein powder in the desired buffer and buffer volume. Moreover, I often observe an additional purification effect through freeze-drying as many contaminating proteins won't resuspend again.

Thanks Jim; this item came up in my search as well; now that you've used it and voted for it; I'm gonna go for it :)

I agree with Jim, Amicon centrifugal filter units are good for concentrating the protein. You can also try Vacuum concentrators if your protein is suitable for it.

I agree with Jim. Amicon centricon are good. I have also used Nanosep from Pall and I was happy with it. The only issue with it is when the protein you have purified is sticky and you might lose some in the UF membrane, so make sure you redissolve the pellet with your buffer and possibly spin the inside cone of the tube upside down to collect all the retentate.

If your volume is more like 20-30mL you can use TFF cassette with low protein binding membrane like PVDF, PES etc. otherwise for smaller volumes centricon are good. However, select the low protein binding ones.

Centricons of particular size range according to you need is the solution to concentrate proteins.

Thanks so much you all :)

Depending on your sample volume, centrifugal filter concentrators from Millipore, Corning, Pierce etc are a good choice. For larger volumes I prefer to dialyse my sample using Pierce's SnakeSkin tubing followed by lyophilisation (freeze-drying). That way you can resuspend your protein powder in the desired buffer and buffer volume. Moreover, I often observe an additional purification effect through freeze-drying as many contaminating proteins won't resuspend again.

Ivelina is right. You should be able to concentrate your enzyme by dialysis aginst a high concentration of PEG. The protein in question is of fairly small size and so be sure to choose a dialysis membrane of suitable pore size. With regard to PEG, manufacturers list a wide variety of products that differ in mol size and solubility in water. Carbowax produced by Dow chemicals is pretty good for removing water. Go to the Dow website and select a variety that is completely soluble. You will need to make a concentrated solution and so solubility is critical. A mol wt of about 500 is probably good but perhaps you can contact Dow for advice. A word of warning. The removal of water can happen fairly rapidly. Therefore keep a watch on the process so that the contents of the dialysis bag do not come completely dry.

Peter Butterworth

@ Ivelina and Peter. What MW cutoff dialysis bags and PEG grade do you use to concentrate proteins?

I commonly use centrifugal diafiltration systems ranging from 0.2 ml up to 50 ml alternatively for bigger volumes TFF cassettes would do the same job.

If you have a large volume, I prefer to use the stirred cell ultrafiltration units (Millipore/Amicon). You can get a 10KDa MWCO membrane. You can also use these for diafiltration if you need to use a different buffer for your reactor and it's much faster/cheaper than dialysis.

It depends how much would you like to concentrate them. We use AMICON system with 10 kDa cutoff. We concentrate them max 5x and it lasts only 5-10 min.

I agree with Mladen.

Why can't we use ammonium sulfate precipitation ? Is the sample too diluted to be precipitated using salting out ? can someone please shed some light on what should be the minimum concentration of a sample which could be concentrated using ammonium sulfate precipitation ?

In using PEG method, there is always some chance of PEG getting into the dialysis bag and contaminating the protein, as it is discussed at many places. Is it possible to remove that trace amount of PEG by dialyzing the concentrated sample against fresh PEG free buffer ?