I am carrying out western blot analysis to determine pro-inflammatory markers in LPS induced murine RAW 264.7 macrophage cell lysates. I am using beta actin as house keeping protein and optimizing fluorescent tagged Ab concentration for the same, I am using Bolt Bis tris 4-12% gels and run electrophoresis at 150V for 35 minutes, with samples loaded at 25 ul/well. Although I got clear and sharp bands at the right place on membrane (42KDa mol. Wt. of beta-actin). I found that there were bands appearing at the top of the membrane where the well bottom surface is. Does this mean that there is still some protein left in the wells, or was the loading volume too high.