- Gel type: Novex 4–12% TG WedgeWell precast gel
- Running conditions: 70 V for 15 minutes (stacking), then 105 V until the dye front reached the bottom
- Protein load: 5 µg per lane
I’m new to Western blotting and need some troubleshooting advice. I’m using Novex 4–12% TG WedgeWell precast gels, and I often see vertical smearing in my SDS-PAGE runs (example image attached). This smearing carries over to my Western blots and makes the results uninterpretable, even though my band of interest is present. I’d really appreciate suggestions on how to reduce or prevent this issue. My protein of interest is smaller than 20kda. I also use fresh running buffer, transfer buffer etc.
Hello Brigette, is it possible that your protein is too close to the running front of the gel? The lower panel in the upper figure strongly suggests this to me. My suggestion for a 20 kD protein would be a 15 % SDS gel or a gradient that ends with 15-20 % acrylamide/bisacrylamide.