Smile Effect on Western Blot Bands (Western Blot (Misshapen / Uneven Bands))

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Accepted Answer

Bands appear curved in a smile-shaped pattern across the gel, with edges migrating faster than the center. This distortion affects all lanes uniformly and compromises molecular weight estimation. Common causes: 1) Voltage too high during electrophoresis causing excessively rapid migration 2) Gel temperature too high during migration altering buffer pH 3) Insufficient cooling during electrophoresis run 4) Heat accumulation in center of gel due to lack of temperature control Solutions: 1) Decrease voltage according to protocol recommendations and gel specifications 2) Run electrophoresis at 4°C on ice or in cold room 3) Pre-chill running buffer before use 4) For large proteins (>100 kDa), run overnight at constant low amperage and reduced voltage