Non-Specific Signal from Insoluble Protein in Well or Dye Front (Western Blot (Unexpected Multiple Bands))

Question

Accepted Answer

Strong signal appears at the top of the gel (in or near the loading well) or at the bottom (dye front), unrelated to the expected molecular weight of the target protein. This is especially common for very high molecular weight proteins (>250 kDa). Common causes: 1) Insoluble proteins above gel separation range (>250 kDa) trapped in loading wells 2) Protein aggregates formed during sample preparation cannot enter gel matrix 3) Dye front transferred to membrane carries non-specifically bound proteins 4) Stacking gel layer transferred along with resolving gel retains trapped material Solutions: 1) Remove stacking gel layer before transferring to eliminate trapped material 2) Excise dye front from gel prior to transfer to prevent non-specific signal 3) Check protein databases for expected molecular weight including all isoforms 4) For high MW proteins (>150 kDa), use lower percentage gels (6-8%) and longer run times 5) Increase sample solubilization by adding 2% SDS and 8 M urea to lysis buffer for difficult proteins