Complete Absence of Western Blot Signal (Western Blot Immunodetection)

Question

Accepted Answer

No protein bands are visible on the blot, even at the expected molecular weight position. Film or imaging system shows no detectable chemiluminescence or fluorescence. Common causes: 1) Primary antibody raised against species different from sample (sequence mismatch) 2) Secondary antibody incompatible with primary antibody host species or immunoglobulin class 3) Primary antibody designed for native protein used on denatured Western blot samples 4) HRP-labeled antibodies used in solutions containing sodium azide inhibitor 5) Antibody concentration far too low to detect target protein 6) Detection reagent sensitivity range does not match target protein abundance Solutions: 1) Verify primary antibody targets the protein sequence from your sample species using NCBI protein database (http://www.ncbi.nlm.nih.gov/protein) 2) Ensure secondary antibody targets the host species of primary antibody and matches IgG class (most common); use phylogenetically distant host from sample species 3) Use antibody raised against denatured antigen for Western blot, or run non-denaturing gel for native protein antibodies 4) Remove all sodium azide from buffers when using HRP-conjugated detection system 5) Increase antibody concentrations systematically (e.g., try 1:500, 1:1000 dilutions for primary) 6) Switch to higher sensitivity detection reagent (e.g., Luminata substrates for chemiluminescence) matching target protein expression level