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trouble in running SDS-PAGE - (Aug/14/2006 )

I run SDS-PAGE to detect a 55Kd protein . First time I use 12% gel that was prepared by someone else and the protein can be seen by western . In the following SDS- PAGE running I prepared gel and buffer but I can't see the protein any more .And the puzzled phenomena was found the first and second band of Multi-colored Standard separated in the sticking gel. When in the separating gel the first and second band stoped and don't separat any more. The gap between them is not more then 0.5cm. Who can tell me why ?

-bush fox-

check the pH of your solutions.
pH6.8 for the stacking, and 8.8 for the separating


Let the gel polymerize for at least 30 minutes. Check, if the solutions are all prepared the right way and you use the running buffer in the right dilution.