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Tricine SDS-PAGE for plant mitochondria - Membrane proteomics (Aug/27/2010 )

I am isolated mitochondria from wheat root. After isolating, try to extract using TCA-acetone methods. Then using Tricine SDS-PAGE (16% and 4%), and running time 30 V for 1 hr, 200 V for 1 hr and 230 V for rest untill reached dye to bottom. Now Come to my problem, show the smearing and amiling effect. Standard marker is so fast than sample, and also make so heat. How can I remove smearing and smiling effect for getting getting good separation.
Please help me.

Abu Hena

-Abu Hena-

smearing is caused by incomplete denaturing of the sample and/or too much sample.

smiling is caused by uneven heating of the gel. to correct this you can reduce the voltage of the run or cool the gel during the run or both.

if the sample is migrating slower than the marker then either the sample is bigger than the marker or the sample buffer pH has been altered by the sample.