SDS PAGElaemelli system modifications if any

[exec]

We have been using SDS PAGE for quite some time for our recombinant protein Mw 20 kdA USING 15% Resolving and 4% stacking and tris glycine SDS Running buffer i.e laemelli system.

 

is there any new methodology for SDS page that will increase resolution and help in getting clear sharp bands, any different buffer system or any such method. we do not want to use precast gels and cast our own gels. Also we use discontinuous Mode of buffer system.

 

Any suggestions are welcome.

 

 

regards

 

 

[CharonY]

Depends really on what problems you have. One could increase the C value, for instance, to further reduce pore size. While it is mostly done for DNA or RNA gels, it may resolve 20 kDa proteins slightly better (at around 12 %). However, the sharpness does not only depends on that. Depending on the chamber you use, several other factors are likely to be more important. Among these heating, SDS depletion and inhomogenous electric fields are very common (again, depending on the system). Counter measures are usually cooling, adjusting the Voltage (or, alternatively, current), run time and buffer reservoir (or use two-buffer chambers), and seal gel caskets (when applicable).

But it really depends on what the issue is in detail (i.e. are the bands diffuse and large?).

Edited July 23, 2011 by CharonY

[WorldOfBiochemistry]

When I want to increase the gel resolution I usually prepare the gel the day before running it. Usually I prepare at room temperature but after polimerization I store it at 4ºC overnight.

 

 

[CharonY]

Yes, complete polymerization and also degassing are good ways to avoid artifacts and gel irregularities (e.g. slightly wavy bands).