The aim is to purify protein by his- tag purification. I assume it requires a protein concentrating step before laoding on affinity column.
Behrooz: You really don't need any special protocol and don't need to concentrate prior to loading to Zn-NTA-agarose column. In practice, we simply add 20 mM imidazole/150 mM NaCl in the conditioned culture medium containing your His-tagged protein, followed by washing with standard buffer with 20 mM imidazole and eluting with 20-250 mM imidazle in the same buffer. Good luck.
In addition to Dr. Zhao's answer above:
- It is not necessary to concentrate the medium. It is advisable to filter it through a 0.22 um cartridge, though. Affinity media tends to be expensive and will get clogged with large volumes of culture medium.
- Yeast culture supernatants tend to be acidic, so you have to ensure that the pH is slightly basic (above 7.5, preferably 8), otherwise the His residues will be protonated and the affinity purification will fail. Sometimes just adding 20 mM imidazole will do the trick, sometimes it won't, depending on the buffering capacity of the culture medium.
Hope it helps!
Dear Drs Zaho and Martin
Thank you very much for your answers. How about 500 ml- 1 L culture medium? isn't it too much to be loaded on columns, which results in keeping the sample in RT for too long? Particularly that I need to assay the protein (enzyme) activity afterwards . Many thanks.
Dear Abir
Thank you very much for your suggestion. I'll get some information on that. I wonder if you have good experience with TPP for example in similar situations to the one I described?
Sorry for the late answer!
Whether 0.5-1L is too much depends on the capacity, maximum flow, etc. of the column. I have pulled a recombinant protein from 5 L Pichia supernatants using a 10 mL Q-Sepharose FF columns so it is doable. Still, you have to account for the residence time of the ligand in the column (even if the column can handle the flow, capture efficiency drops when residence time becomes too short).
About your sample requirements regarding temperature: Can you not borrow a jacketed column (i.e. GE's XK low-pressure series) and pump chilled water through the jacket, while keeping the sample reservoirs on crushed ice?
Hope it helps!
Good suggestions Alejandro, I'll start with lower volumes first and then make decision on how to follow based on what I get. Sometimes less is more! Thanks very much.
Hi Andrea
Thanks very much for sharing your experience. I work with S. cerevisiae.
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