Streaking and Uneven Migration from Buffer Issues (Western Blot (Misshapen / Uneven Bands))

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Bands show streaking, smearing, or uneven migration patterns. Multiple bands may merge or appear poorly resolved with inconsistent migration across the gel. Common causes: 1) Incompatible sample buffer composition affecting protein charge or solubility 2) Inadequate salt concentration in lysis buffer causing poor protein migration 3) Insufficient detergent (SDS) concentration in sample buffer 4) Incorrect sample buffer to lysate ratio during preparation 5) pH variation in sample preparation buffers Solutions: 1) Verify adequate salt concentration in lysis buffer (typically 150 mM NaCl) 2) Ensure proper detergent concentration (standard 1-2% SDS in sample buffer) 3) Use fresh sample buffer with correct composition (standard Laemmli buffer: 62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol) 4) Maintain consistent sample buffer to lysate ratio (typically 1:4 v/v) 5) Heat samples at 95-100°C for 5 minutes to ensure complete denaturation