I would like to perform a native PAGE of my small protein ~6.5 kDa to see its physiological oligomeric state. Normally, I use tricine SDS-PAGE to separate the proteins but I am not sure whether there exists a native PAGE using tricine gels or not?
Thanks in advance for all the suggestions.
Although I don't know for sure, I strongly believe that even if there is no protocol available, you could do it yourself successfully. What I mean is that I see no problem on running a Native PAGE using Tricine. Running buffers contribute to the PAGE technique as the separating "force", both conducting current and establishing the pace at which migration occurs. Being native or denaturing (SDS) differs only in chemical composition of the environment regarding denaturing agents, and is defined mostly by gel-content in SDS and sample treatment prior to loading.
Obviously, in an attempt to do it, SDS of the Tricine-buffer would have to be discarded from the recipe.
The reason it might be odd to find this specific protocol is that the needs of someone using native and someone using tricine are usually opposites: in native, because we're looking for oligomers or big 3D conformational states, we are not worried about the low molecular wheight zone of the gel. On the other hand, someone using tricine is looking for small peptides, in which case denaturation might be necessary to mass-resolve, free from oligomers. An oligomer of low molecular wheight (at least so low it won't be seen by glicine/bicine) is not very common, and for that reason the technique PAGE mixing native and tricine might not have been explored yet.
Although I don't know for sure, I strongly believe that even if there is no protocol available, you could do it yourself successfully. What I mean is that I see no problem on running a Native PAGE using Tricine. Running buffers contribute to the PAGE technique as the separating "force", both conducting current and establishing the pace at which migration occurs. Being native or denaturing (SDS) differs only in chemical composition of the environment regarding denaturing agents, and is defined mostly by gel-content in SDS and sample treatment prior to loading.
Obviously, in an attempt to do it, SDS of the Tricine-buffer would have to be discarded from the recipe.
The reason it might be odd to find this specific protocol is that the needs of someone using native and someone using tricine are usually opposites: in native, because we're looking for oligomers or big 3D conformational states, we are not worried about the low molecular wheight zone of the gel. On the other hand, someone using tricine is looking for small peptides, in which case denaturation might be necessary to mass-resolve, free from oligomers. An oligomer of low molecular wheight (at least so low it won't be seen by glicine/bicine) is not very common, and for that reason the technique PAGE mixing native and tricine might not have been explored yet.
Thank you for the explanation, the answer is very clear in itself!
I agree with Rafael's suggestion to run the gel with the same bufer system, but leaving out denaturant. Yet, in this case, the migration will depend not only on the molecular mass but on the net charge of the species to be analyzed. In order to take this into account, gel electrophoresis should be performed at different polyacrylamide concentrations and the results analyzed using Ferguson plots
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