I am using G-bioscience C-18 spin column for peptide desalting, following vendor's instructions. I tried several times but I lost most of peptides in bonding flow through. I also tried remove organic solvent in the sample buffer for better binding. I found some paper in which people used waters Sep-Pak C-18 cartridge. I am wondering the difference between the two. Can anybody suggest some suppliers of the cartridges or spin column with good peptide recovery or do you have other insights or suggestions?
Thanks a million for your help in advance
The C18 acts the same in a spin-column, a sep-pack cartridge, or a column, and all can be used for desalting.
Knowing little about your particular peptide and how you prepared the sample and cartridge, possible problems could be:
Read and follow the spin column instructions.
Hi Yongling, I second what Jack said in his answer. And I would add: check pH of the buffers and sample before loading to the columns/cartridges. For classic C18 desalting, pH should be bellow 4. As already mentioned get rid of organic solvent in your sample. I usually dry the sample in SpeedVac and resuspend in the loading buffer (something like 2% ACN, 0.1% TFA in water). I have good experience with SepPak cartridges and Thermo C18 spin columns.
Hi Jack and Lukas, thank both of you very much for your nice suggestions!!
1. I guess the difference between spin column and waters sep-pack cartridge might be the peptide capacity.
2. I have solved the peptide loss issue and I am sharing my experience here with other people. Previously, I just followed the vendor's protocol using 50% Methanol as the conditioning solution. However, when I used 100% Methanol to condition and wet the C-18 spin column, I got a very good peptide recovery tested by mass spec. So I agree with you that the first wetting step before the sample loading is very important and ensures the interaction between the peptide and C-18. I am also confused why the protocol from the vendor didn't work.
3. I used 0.5% FA in H2O as the equilibration solution and the solvent to re-dissolve my peptide before sample loading. One thing I am concerned is that if this would cause phase collapse (deactivate) the C-18 hydrophobic chain. I also found some peptides they used 100% H2O as wash solution.
4 The peptide I tested is WAGGDASGE which should be easily retained on C-18.
5. Yes, I cheked the pH ~2 before loading the sample.
Thanks again for your useful suggestions! Really appreciate it. Jack Silver Lukas Krasny