Denaturing Gels and non denaturing gels (Gel electrophoresis) - Gel electrophoresis (Mar/29/2008 )
When I use a non denaturing gel and I also want to use a stacking gel, then should I place the stackinggel in the middle of my running gel?
Or should I place the stacking gel just at the top of my system? (just underneath the buffersystem?)
Someone told me I can just put the stackinggel underneath the buffersystem (like you always do when using a denaturing gel) , but when I do this, then I think I would not get a good seperation based on electrik charge because the negative pole (in the buffersystem at the top) is right above the stackinggel and thus meaning I would not get a good separation on charge.
I mean: when I put my proteins in the stackinggel and this stackinggel is placed right underneat the buffersystem at the top wich is - , then the + charged proteins will move to that buffersystem and thus not be separated good because there is almost no running gel between the stackinggel and the (-) bufferstystem at the top.
I know that when using sds all the proteins will be negative so they will all move towards the + buffersystem wich is placed at the bottom so I can easly put my stacking gel at the top, there is no protein that is + charged so they will not move towards the top buffersystem wich is - charged.
Or are stackinggels never used when using a non denaturing system?
in native page you are still sending your protein through in one direction. you manipulate the pH such that your protein of interest has a charge that will migrate through the gel (i didn't specify + or - because that would depend on the pH of the buffer system and the pI of your protein).
in standard native page, the buffer system is basic. since many (if not most) proteins have a pI in the range of 4-6, they will be negatively charged and will migrate towards the + electrode.
there may still be some proteins with positive charges in this system but they will not be resolved. if you want to resolve them then you will have to change the system.
so, in answer to your question, you always put the stacking gel on top of the running gel (although your thought about putting it in the middle is provoking, it would be difficult to introduce the sample into the stack between the plates).
Ah I see,
thanks a lot mdfenko