First, look at your target band. It should be a sharp, horizontal line at the correct molecular weight. If it looks like a banana, your gel was too hot. If it’s a smiley face, your gel was too cold. Sorry, you can’t fix it now—just curse softly and move on.
Now, ignore the black hole at the top of the membrane. That’s probably unresolved high-molecular-weight stuff—your lysate being a messy drama queen. Don’t panic. Focus only on the region around your target (e.g., 50 kDa for tubulin). Everything else is noise, literally.
What about double bands? Sometimes you get two lines close together. This can mean phosphorylated vs. non-phosphorylated forms, or a degradation product. Check your literature. If the doublet is exactly twice the weight? That’s a dimer, not a ghost. Unless it’s your project’s ghost.
