Extra Bands at 50 kDa and 25 kDa Masking Target (IP-Western Blot)

Question

Accepted Answer

Western blot shows strong bands at approximately 50 kDa (heavy chain) and 25 kDa (light chain) that may mask or interfere with the target protein band of interest. Common causes: 1) Heavy and light chains of the primary capture antibody are being detected by the HRP-conjugated secondary antibody during Western blot 2) Secondary antibody recognizes denatured immunoglobulins released during SDS-PAGE sample preparation 3) Capture antibody chains co-elute with target protein from immunoprecipitation beads Solutions: 1) Use a secondary antibody that specifically detects native (non-denatured) immunoglobulins only, avoiding recognition of denatured antibody chains 2) Use crosslinking IP method by first crosslinking protein A or protein G beads to the capture antibody using DSS (disuccinimidyl suberate) before adding lysate 3) In crosslinking IP, the heavy and light chains remain covalently bound to beads and are excluded from the eluted sample