I am having trouble separating my small peptides OR peptides with a mass range of 1–100 kDa using the SDS page gel electrophoresis at 10% resolving gel and 5% stacking gel.
Is there any preferred electrophoretic system for the resolution of small proteins?
Recommendations are highly appreciated.
Thanks
Tricine sds page works for small peptides. I have used it to resolve peptides down to 2 kDa
Increase the gel concentration to around 20%. The main issue is pore size through the gel. However, when you get too small in peptides the SDS binding ratio may change and not all the peptides will be uniform in net charge. The velocity in electrophoretic separations is based on the mass-to-charge ratio. SDS binds with a hard negative charge in a uniform ratio 1.4:1 to most proteins, but at the peptide level this can deviate a lot. Therefore, you can't trust the MW sieving ratio of the gel. You might be better off running a high concentration native gel with an extremely low or extremely high pH buffer, and then confirming the MW and identity of the peptides via MS. If you add SDS, its hard to run MS. Of course without the SDS you have a staining problem, so you may have to run two gels side by side staining one and cutting the corresponding bands from the other.
Tricine-PAGE would be ok for this. Do not forget to fix the low molecular weight proteins/peptides into the gel using glutaraldehyde solution after the SDS removal and prior to coomassie or silver staining for detection...
I've had extremely positive results separating proteins ranging from 4-7 kDa using Tricine SDS-PAGE.
Be aware that it will take significantly longer, though (around 5-8 hours).
There is also a special protocol by Schägger (the original inventor) on how to silver stain and blot these gels.
Regular coomassie staining works just fine.
Another thing to be aware of is to cool the Gel during electrophoresis. Due to the high voltages and prolonged running time, the gel can get quite hot, but it shouldn't be any higher than 40°C. I personally do all my Tricine PAGE in a cold room.
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