When I pour a stacking gel on top of the resolving gel, and run the protein sample, and ladder, the top half of the resolving gel has no marker separation. It is either absent or it is a streak. I have checked the pH of the stacking buffer, its fine, 6.8. Can anyone help on why the presence of stacking gel is causing the problem?
(when i run the same with only resolving, ladder seems to run fine)
ionic strength changes between stacking and resolving gel play crucial role in protein electrophoresis, did you check the pH of resolving gel and running buffer. what's the molarity of of buffer used in resolving and stacking gel preparation. what is the seperation position (distance from top) of ladder when run without stacking. it is the same to stacking.
http://products.invitrogen.com/ivgn/product/NP0323BOX?ICID=search-product
Neutral pH gel is better than traditional method.
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