Insufficient Protein Sample Loading (Western Blot (Weak / No Signal))

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Western blot shows weak or no signal for target protein. Loading controls may also appear faint, indicating insufficient total protein loaded per lane. This is particularly problematic for low-abundance target proteins. Common causes: 1) Total protein loaded per lane below detection threshold (typically <5 µg) 2) Target protein expression level too low relative to sample amount 3) Protein degradation during sample preparation reducing available target 4) Inaccurate protein quantification leading to underloading Solutions: 1) Increase protein loading amount (typically 10-50 µg total protein per lane, optimize for target) 2) Concentrate samples using ultrafiltration or precipitation methods before loading 3) Re-quantify protein concentration using Bradford, BCA, or Lowry assay 4) Add protease inhibitors to lysis buffer and keep samples on ice during preparation 5) For low-abundance proteins, load maximum amount compatible with gel resolution (up to 100 µg)