I'm relatively new to western blots and have been running them for the past few weeks but today, after running 2 gels simultaneously, I got an image with two distinct band layers from a single antibody. Here are the details:
Two gels were prepped simultaneously with same concentrations of running and stacking gels. After polymerizing, one running gel had an unwanted indent, so only half the gel was loaded to avoid indented area.
9 and 4 samples + standard were loaded onto each gel, with several samples present in both gels.
Transfer was performed at 400mW for 40 minutes each. However, one gel ended up with sizing issues, and might have possibly dried out more. Power was 23W as opposed to normal 5-13 in other gel, and gel overheated. PVDF began to dry out, but was immediately immersed in liquid.
PonceauS on overheated PVDF only showed one distinct though messy band layer.
Primary and secondary antibodies were made in same solution and aliquoted evenly to each PVDF. ECL amts were same as well.
Now, the 4 sample gel was imaged after ECL and produced a single clean, normal band layer. The image from the overheated gel produced the expected messy band seen on the PonceauS as well as a second distinct and even band a significant distance above the expected band. The expression varied per sample between the two band layers, but both had similar intensity on average.
Could overheating have caused a second distinct band layer to form? Is overheating known to distort samples in this way? Or is it possible that the greater power might have pulled more sample onto the PVDF that was able to bind to the antibodies? I would appreciate your advice!