I encountered a problem during electrophoresis where I found that the bands were not evenly aligned during protein separation (after migration into separating gel). I also used freshly prepared running buffer. I used 10% separating gel (100V, 10 mins) and 4% stacking gel (150V, 60 mins). I load 50 ug of protein into each well. Is there any tips that I can use to improve the formation of the bands? Attached herewith is the photo I have taken during electrophesis.
correction: I used
4% stacking gel (100V, 10 mins)
10% separating gel (150V, 60 mins)
Keeping the temperature constant across the gel is important. Use the recommended filling of the upper and lower chambers with running buffer. If you are using the Bio-Rad minigel apparatus with one cassette in use, you need 800 ml of running buffer. Fill the top chamber all the way up and make sure there is no leakage. The rest of the buffer goes in the bottom chamber.
We faced such problem and have solved it by running the stacking gel on 50 V for 25 to 30 minutes.
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