To purify the protein, I am using 0.5% Triton X-100 because it is hydrophobic. Lysis buffer has ne contain imidazole and elution buffer has 250 mM imidazole. I tried to optimize imidazole concentration as 0 mM, 20 mM and 40 mM. For 0 mM concentration, I could not obtain purified protein and for 20 mM and 40 mM imidazole concentration, I lost the protein in column wash after sample loading. How can I solve this problem?
Thank you.
Dear Melis Yalçın
just to questions:
1) for hydrofobic do you mean a membrane protein?
If yes i think that you have to optimize the detergents since is possible that in 0,5% Triton X the protein is aggregate and the His tag are buried into the aggregate and so the binding is very weak.
Most commmonly used detergents for membrane protein purification are DM, DDM; OG, which are much more expensive than tritonX but with properties and micelles size more appropriate for membrane protein purification
Article An Overview of the Top Ten Detergents Used for Membrane Prot...
2) Are you using a 6X or 10X his tag? and the tag is directly connected with the folded domain of do you had some linker between tag and GOI?
Because if you are using a 6X His with out any spacer/linker shift to a 10X HIS with a linker (es the TEV recognition site, or a GSGGG linker) could be a possiblle way to improve the tag exposure and the binding on the resin in case your protein is not fully aggregated. In this case before you need to solve the problem on the point 1.
good luck
Manuele
Manuele Martinelli Thank you for your answer.
1)Yes, it is membrane protein. However, if it is aggregate with triton, I could not see on SDS-PAGE. I can see band for protein in column wash on SDS-PAGE.
2) I'm using 6X His tag.
Dear Melis Yalçın
I tihnk that you have to try different detergents.
When you run the sample in SDS page, do not denaturate at 95°C because the high temperatures can induce the aggregation of mermbrane proteins but at lower tenperatures /eg 60°C or 70°C )
best regards
Manuele
Can you able to confirm tagging after expression by using immunoblot-dot blot/ELISA using an anti-his tag antibody or mass spec rather than PAGE?
On the other hand, you may use Co-NTA IMAC for a high binding affinity alternative...
You may validate the IMAC column also using another his-tagged protein as positive control if exist. Stripping and regeneration is needed sometimes for efficient binding if the cartridge is previously used for other purposes.
Increasing or modifying detergent composition would be another point to be discussed.
Good luck.
Emir...
Dear Manuele Martinelli
After denaturation at 95oC, I can see on SDS, I think it is not aggregate. I lost the protein in column wash during affinity chromatography.
If I can increase or decrease Triton concentration, what you think effect on protein? Is it effective to solve this problem?
Dear İsmail Emir Akyildiz
I cannot confirm.
I use Co-NTA IMAC also, still I lost the protein. I know that IMAC column works effective, there is no problem in column.
What do you think about which highest detergent concentration which not harmful to the protein?
Best regards.
Nonionic detergents (Triton, Tween, NP-40, etc.) Removes background proteins and nucleic acids Up to 2% can be used. Cationic detergents Up to 1% can be used. CHAPS Up to 1% can be used. Anionic detergents (SDS, sarkosyl) are Not recommended, but up to 0.3% have been used successfully in some cases...
https://www.qiagen.com/it/resources/faq?id=9c412fe7-a0c9-4ed4-a824-cb7d89895c41&lang=en
9/1/22
Dear Melis,
You said that you used 0 mM, 20 mM and 40 mM imidazole with our affinity column. It looks like you were using a step gradient. If your protein was removed @ 20 mM imidazole, it looks like it binds weakly to your column. You might try running a linear gradient instead, e.g., 0 to 15-20 mM imidazole. Another option is to change the metal on the column. We used IMAC to do affinity chromatography of a His-tagged protein. In our case, the IMAC column had Ni instead of Co. This might improve the binding by your protein.
I hope these suggestions help you.
Bill Colonna Iowa State University, Ames, IA, USA, [email protected]
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