Hi everyone!
I am purifying a peptide from a mixture of proteins. The peptide (3 kDA) is in a solution containing a high concentration of urea and imidazole. I would like to be able to change the medium to a saline solution (like PBS or NaCl solution).
What would you recommend me to change the medium?
I am currently working on a laboratory scale.
Best Regards
Use tangential flow filtration (TFF).
Tangential Flow Filtration - Laboratory | Pall Corporation
There is dialysis tubing with a 1000 Da or 2000 Da molecular weight cutoff. That would, in principle, retain the peptide while allowing imidazole and urea to pass through. It may take a few days. Dialyze against the buffer you want the peptide to end up in, and exchange the bath as needed to dilute the urea and imidazole sufficiently.
Dear Benjamin
you can use several methods. One as suggested by Adam is certanilly the dialisys, however my prefered buffer exchange approach for small volume is desalting, which is very fast.
It is a sort of Size exclusion chromatography based on coloumn with very low exclusion limit. In this way buffers entering into the coloumn and run slower than the protein or peptides those are going in to the dead volume.
For small volume (up to 2-2.5 ml of sample) GE offer 2 different kind of columns
for peptides (MW >700Da) the PD-mini e PD-midi trap
28926749.pdf (cytivalifesciences.co.jp)
for protein (MW>5Kda) the PD-10 coloumns
PD-10 desalting columns packed with Sephadex G-25 resin | Cytiva, formerly GE Healthcare Life Sciences (cytivalifesciences.com)
i''m routinelly using the second for buffer exchange with protein. I have limited experience with the fist but i do not see any reason why it has to do not work well as the PD-10.
To perform this process in important that you have in your hand a tool to detect the peptide in the eluted fraction and that the peptide concentration is enough to provide a detectable signal. Eg: For protein i'm using the bradford assay of UV-vis 280nm.
If you are interested to see more detail about desalting you can see the following video
1) buffer exchange method overwiev (https://www.blogger.com/video.g?token=AD6v5dzeX21U_Er6jBChQIhE5zWtQhNjRjNXRPR7qk7KfWWSQxhf8PbUn4R6KLL0pn_7tU92M0-B3kEUHO9IkTpelmdO79QR53uSg29ZFV3j5D-siHAamTu1y2wOEQ8S4As_wpxzLvA)
2)https://www.blogger.com/video.g?token=AD6v5dwNlVHz3BclDT9RLY-6KbEEqkS3RnRdHn4BqTsZW4j_prf3KZwHDMTzRYT-B9Yifvmde7RdExL5AArLFtg1Q13fu4-83o3M8bCka1jJ5PfWFK8B53BOpx6YrzvLr2qDiPgT15c
rapid buffer exchange by PD-10 avaialble on my blog ProteoCool
Use a small pore size (e.g.100-120 Å) Reverse phase peptide mapping column and get rid of proteins but also the ionizable excess solubilization molecules such as imidazole and urea. Increasing ACN can elute your peptide in a small amount if your peptide is not very hydrophobic. Use speedvac lyophilisation or alternative approach to evaporate the organic moiety and resuspend your peptide with desired buffer media.
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