Hi all,
I have run BN PAGE twice so far and am seeing distortion of the bands at the bottom of the gel.
I use a 4-15% precast gradient gel. I load 15-20 ul of sample in every other well and overlay with 15 ul stacking gel buffer (75 mM imidazole, 1.5 M 6-AC). Empty lanes are filled with the same volume of stacking gel. The inner chamber is filled with a cathode buffer (50 mM tricine, 7.5 mM imidazole, 0.02% coomassie blue), and outer chamber with anode buffer (25 mM imidazole). I run at 100V, 4 degrees C, and allow the sample to enter the separating gel for about ten mins, then switch to 200V. Once the samples reach the bottom in about 45 mins, I terminate the run.
Please take a look at the attached photo. Ignore the leaking of sample into adjacent wells; this is an issue I've fixed. Thanks in advance for your help.
it looks like a small layer of air has got trapped at the bottom of the plate in 2 places and there is no continuity between the buffer and the gel so near the bottom of the gel the electric field is contorted around the air
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