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This frustrating (and common) problem can ruin days of work, but the fix is often simple. Our comprehensive troubleshooting guide diagnoses the real reason for your patchy bands. We cover everything from the #1 culprit—air bubbles in your transfer sandwich—to aggregated antibodies, uneven gel polymerization, and improper sample prep. Stop guessing and start fixing. This article provides clear, actionable solutions to rescue your blot and get the clean, sharp bands you need.

Is your Western Blot showing a band at the wrong molecular weight? This is one of the most common frustrations in the lab, but it's not a dead end. An unexpected band size is often a critical clue, not a failed experiment. The theoretical calculated weight from amino acid sequence rarely matches the observed weight on a gel. This discrepancy can be caused by a host of biological reasons from post-translational modifications like glycosylation to protein cleavage, splice varia