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Concentrating Protein - 8M Urea, Protein solution, concentrating (Nov/02/2009 )

Hi there,

very much often I have to purify under denaturing conditions using 8M Urea. To proceed with the next step I have to dialyze the sample in the next step and concentrate the protein solution. Usually I took the Vivaspin20 columns what is an excellent solution. But now my samples range in the size of around 100-300 ml what is quite a problem using Vivaspin. First Vivaspin works not that good with Urea because it takes a long time to just concentrate 20 ml. Second the volume is too large that by applying the whole onto the spin column the membrane could take harm of reusing it >5 times. Taking many columns also is quite expensive. Is there another method you could suggest to me to concentrate proteins from Urea solutions?

What else I could think of:

- dialyse first => concentrate using PEG or lyophilisation

thanks in advance :)


After the dialysis Maybe acetone precipitation ? Just wondering if then your protein will still be soluble in aqueous buffer ?


100-300 ml of 8M urea is a very large volume to work with! Can you not work in smaller volumes?

Does your protein refold nicely? Once refolded, how do you purify?


you can use an ultrafiltration cell such as the amicon (now from millipore). you can concentrate large volumes and can also exchange buffers (diafiltration).


I fish the protein from the cleared cell lysate using a histrap column. The concentration of the proteins in solution from destroyed cells is quite high so I can not load so much onto the column. So I run a method using the ÄKTA Purifier and concentrate the eluate fractions afterwards. Per run the target protein remains in 6 ml eluate resulting in 10-20x 6ml. During the day its not a big deal to use centricons/vivaspins but after a weekend it just takes ages to concentrate these elution fractions. The amount of protein I gain is very little so I need a lot of cells to produce it (organism / conditions / etc). Thats why a lot of other proteins also bind to the column. Another point is that the protein elutes also very early from the column so I can not increase the amount of imidazole in buffer A to get rid of unwanted protein contaminatants. This all results in repeating the purification for some time what is quite time consuming. Concentrating the protein from the destroyed cells may not be the best idea I think because than I would just overload the column. Maybe you have an other idea about my strategy that could help.......


Can you give us the details of your washing and elution?
Also, you could process more lysate with loose beads in a batch adsorption protocol, or daisy-chain smaller columns to give greater capacity.
Can you modify the construct to have 10 His residues, to increase the binding affinity? What changes to the binding and washing buffers have you tried?


what is your next step? you may not need to concentrate.


It sounds like you are using the HiTrap column from GE. If so, are you using the HisTrap™ FF crude and what size column. The product spec. states 40mg/mL resign and even for a conservative estimate of 20 mg/mL sounds like a lot of binding capacity. This is meant for purifying from a crude extract. As was mentioned by Swanny, you could attach several columns together.
As far as concentrating, the Amicon stir cells mentioned by Mdfenko are good for your needs, but you can also look at the Jumbosep Centrifugal Devices from Pall (Found in VWR). These hold 60 mLs per cup and a full centrifuge would be 360 mLs. Just make sure you have a centrifuge that has the right inserts for these.


I am new to handle intricate protein purification, now going through a similar problem that is mentioned above in concentrating my protein. I do His-trap -> dialysis -> DEAE-trap -> dialysis at the end I had something like 10 ml of dialyzed protein at a conc. of 0.17mg/ml. For the enzymatic assays I planned to do it would be much better if the conc. is at least 2mg/ml. With PEG 35000 I already reduced the volume to 3 ml. I am just too scared to leave it in dialysis bag just because one can't see the volume reduction that clear in them. I transferred the contents from the dialysis bag to 3 eppis and tied their mouth with dialysis membrane, leaving the tubes upside down on a bed of dry PEG. I see the volume continue to reduce but at very slower rate, mean while the membrane is also getting dried.

now my worries are:

1. Will the concentrating process be still effective like this.

2. Is there any other better ways (except amicon, acetone precipitation- I just don't want to loose protein in those, I am reading a lot of issues around them here in bioforum, and lyophilizer- as we don't have a refrigerated one) to concentrate protein without loosing its native activity.

3. All the processes I mentioned above to extract and purify my protein took 1 week, since the day 1 they are in 4 degrees, and I don't know when this concentrating process will end. How much likely that I loose my protein for degradation, and to what extent.

condition I think I can't change: is the initial volume of cell lysate. I already start with a viscous extract even after DNase treatment.

Thanks in advance!