1. Large gel 16%6M urea
2. Mini gel 16%3M Urea, silver staining
3. Mini gel 18%4Murea
Fixing: 50%methanol, 10%acetic acid, 100mm ammonium acetate
Staining: Coomasssie
Destaining: 10%acetic acid
voltage:30V in the first step, 90 V in the next step
The method looks good. Make sure you try the low weight markers. Make a run as a control. If it doesn't work then there is a problem. Otherwise repeat with new materials, check the expiry dates
I think there is need to improve gel preparation, loading and running processes as looking in your band. Try to add amount of 4μg proteins per well for all Tricine-SDS gels, also try with 60V in first step and maintained at this voltage until the samples completely entered the lower separating gel. The next voltage steps were set at 100V for 1.0mm 10%T gel and 140V for 1.0mm 18%T gel.
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