I am working with an initial volume of 20 mL* and using a 10 kDa filter to separate proteins from peptides. With this filter I am able to concentrate the proteins upstream of the membrane. However, I have still the peptides that I am interested in analyzing by Mass Spect in the filtrate (around 20 mL). I am thinking on the possibility to use another 1 kDa filter to concentrate them (it is the smallest I've found), but I am worried to lose some of the peptides with low MW. Any recommendation?
*The extraction buffer includes urea.
You can use solid phase extraction and also get rid of urea. I would use reverse phase adsorbents.
I normally discourage everyone from using gel filtration, but your application would be perfect. Run the protein/peptide mix over a gel filtration resin like Sephadex 25, with a MWCO as low as possible The proteins should just race through the column and your peptides elute with the salts, etc.
You should use a high concentration of urea or guanidine HCl in your sample at least; as running buffer you could try one without any chaotrope.
If you don't have an HPLC and columns for reversed phase chromatography, you can simply use Solid Phase Extraction spin columns and elute with a smaller volume of ddH2O.
http://www.thermoscientific.com/en/product/hypersep-c18-spe-cartridges.html
Thanks Ladan Ajdanian . I looked into these columns yesterday and I have emailed Thermo to figure it out more details about them. Do you know if I need a specific vacuum system to use them?
Dear Ana Gil de Bona
I think you do not have any specific vacuum system, but when I know about this one, I will send you a message very soon.
Best Regards
Dear Ana Gil de Bona
I think this paper can help you.
Article Comparison of ultrafiltration units for proteomic and N-glyc...
Best Regard
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