You can try PD-10 disposable columns from Amersham (now GE Healthcare) for desalting of your protein by SEC. It's fast, it's reliable and it's not expensive. We have been using this column for many years to separate the bound radioactive I-125 from the unbound fraction and never had any problem.

You could use trichloroacetic acid (TCA) to precipitate the proteins. This method is suitable for preparing protein samples for SDS-PAGE, even if they are dilute or in urea. The procedure I have used is to add 1/10 volume of 72% (w/v) TCA and 1/10 volume of 0.15% (w/v) sodium deoxycholate. Store the samples on ice for at least 10 minutes, then spin at 16,000 x g for 10 minutes. Remove the supernatant thoroughy, being very careful to avoid removing the pellet. Add 1X SDS-PAGE sample buffer to dissolve the pellets.

I would try TCA as indicated by Adam B Shapiro. Note that TCA can cause the pH of the SDS-page sample buffer to drop (you can see this by a change in the color of the sample buffer from blue to yellow). Therefore, sometimes the precipitation is followed by washing the pellet using ice-cold acetone to remove traces of TCA. You have to completely resuspend the pellet in ice-cold acetone by vortexing, followed by another round of centrifugation. If necessary, you can repeat this step a few times. Finally, you should allow the acetone to evaporate, and then you can dissolve the dry pellet in 1x SDS-page sample buffer.

There might be some issues if you want to use SEC to tackle your problem. Although PD-10 columns are great for changing the buffer and/or desalting your sample, I would strongly advise against using SEC in this case. You indicate that your aim is to remove the salts and urea, which implies exchanging to a urea-free buffer. Often, protein will aggregate on the column during separation from urea. Furthermore, if you have many samples, and/or small sample volumes, the PD-10 columns are quite inefficient because of the time it takes to run them.

I agree to both, either try precipitation (cheap and quick) or a centrifuge column (Amicon/Centricon or similar, also quick but more expensive than TCA).

Thank you all for your quick responses! I'm trying to make thin films/membranes out of this protein (so it is going to be treated with 90% formic after precipitation after I precipitate it). The salts (especially urea effect phase separation and assembly of the protein). Ideally This should be a quick method of precipitation (as dialysis works but it is too time consuming for the work I am undergoing). The sample is only being used for thin film production as this is the next stage of my project (SDS-PAGE will only be used to indicate purity up until the point of thin film production>

Thanks again for your quick responses. I really like the idea of both but unfortunately I do not start my PhD for a few months and it is mainly on the materials side, so hoping to resolve production before then. Also, waiting for funding so the cheapest/easiest way possible would be best for me at the moment.

Thanks again!

The gentlest, easiest way to precipitate proteins differentially from solution is with saturated ammonium sulfate, but not all proteins respond. If yours does-- very fast, cheap and you can store in it for some time before further processing.

But skipping dialysis may be a false economy of your time-- this is the most critical point in terms of yield, purity and contaminants in your procedure. Make peace with it, buy a set of clips and some tubing-- very inexpensive, not all that time-consuming, and far better material without nasty surprises later that are hard to explain.

Quantitative protein precipitation is in my experience best achieved with chloroform/methanol precipitation (doi:10.1016/0003-2697(84)90782-6), this has a better recovery than the DOC/TCA method.

You can use PD 10 columns to remove salt and urea. This is not very expensive and save your time.

I would absolutely START by using Dialysis, with large volume 1-5L of buffer (and glycerol if you want to freeze it away). What I generally do is dialysis while slowly lowering the Molarity of the Urea. I start with 8M, then 7, then 6, ect. waiting at least 4 hours. Finally making a buffer with zero Urea, with 5-10% glycerol, and the buffer of your choice.

Sir Engelbert Buxbaum ,

I would like to know what are the suitable buffer to dissolve the protein pellet instead of 5% SDS? Im using Bradford assays instead of Lowry.

• You could use an alkaline solvent (dilute Na2CO3 or NaOH) and then the BCA assay, which uses alkaline conditions anyway.
• Alternatively, you could hydrolyse the protein pellet with Ba(OH)2 and then determine amino acids with ninhydrin or OPA (see doi:10.1016/0003-2697(72)90245-X or doi:10.1006/abio.1996.0503).
• Proteins dissolved in SDS sample buffer can be determined by filtration over hydrophobic membranes according to doi:10.1006/abio.1994.1595 or doi:10.1016/S0003-2697(03)00255-0