Is there any special centrifuge filter or another instrument for the purpose of tiny volumes?
Easy: There are a lot of dialysis filter devices that can be spun in centrifuges. Amicon/Millipore is a good provider of nicely working centrifugal filters. They are, however, nicely expensive, too.
Easy: There are a lot of dialysis filter devices that can be spun in centrifuges. Amicon/Millipore is a good provider of nicely working centrifugal filters. They are, however, nicely expensive, too.
Dear Johannes, Thanks for your answer. In fact, the problems is with millipore filters. we have them in our lab. but the needed volume is 10 ml . My current volume is 0.4 ml. Is it possible to fill the millipore filter with 0.4 ml?
There are smaller ones, essentially Eppendorf tube style. Tomorrow, with a faster net connection, I can give you details.
Hi Oveis,
I have used the ultrafilter tubes of Millipore. Attached is the instruction of the tubes-0.5ml. Hope it will help. Good luck.
Regards,
Xiaojing Sui
These should meet your requirements: Amicon Ultra-0.5 mL Centrifugal Filters for Protein Purification and Concentration. Click on http://www.millipore.com/catalogue/module/c82301 for detals. Xiaojing Sui's link provides the necessary instructions.
Dear Xiaojing,
Thanks for your help. I should search for such facility in our lab.
I would like to recommend the UPPA Concentrate Kit Kit from Geno Technology, Inc., St. Louis, MO, USA, in particular for samples with very low protein concentration.
You can also dry your samples down in a speed vac (not all the way to powder, but almost), depending on what buffer(s) they are in and then resuspend in a more concentrated buffer using less volume.
I support Johannes' suggestion about using Amicon Ultra. I used it to concentrate histone octamer and to concentrate/desalt transcription elongation complexes. Be aware that some proteins get absorbed on the membrane. Adding BSA to 0.2 mg/ml final fully eliminates all the losses on the membrane, but you can not always afford adding BSA to your precious purified protein. If you need a pure protein, test run with 10% of your prep, and check how much is lost.
As mentioned by Maria, some proteins are significantly absorbed by Amicon membranes. We also observed that the desalting and concentration process can be very long for high volumes at low temperature (4 degrees) using the Amicon/Millipore systems, up to one working day. If you are working in the neighborhood of chemists, an unusual and alternative solution would be to use HPLC for the last purification step. The main advantages are that you get your protein highly pure and fully desalted after lyophilization. You then can adjust the concentration of your sample by simply resuspending the pure powder in the buffer you want. However, you need to ensure that your protein will not precipitate in the presence of organic buffers and inside the column. I used to think that HPLC is not suitable for last step purification of proteins but I've changed my mind since we were successful with distinct proteins.
Depending on volume: can always use dialysis tubing and polyethylene glycol (PEG).
use ammonium sulfate precipitation and then resuspend your precipitate in a small volume and make a dialysis unit using an Eppendorf cap covered with a dialysis bag
What could be the appropriate pore size of the membrane filter to elute out 150-160kD protein
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