Complete Absence of Signal in Western Blot (Western Blot Immunodetection)

Question

Accepted Answer

No bands are visible on the Western blot membrane or film. Complete absence of signal despite confirmed protein loading and transfer. Common causes: 1) Primary antibody raised against different species sequence with no cross-reactivity 2) Secondary antibody host species incompatible with primary antibody (e.g., both raised in rabbit) 3) Primary and secondary antibody class mismatch (e.g., IgG primary with IgM-specific secondary) 4) HRP-conjugated antibodies used in solutions containing sodium azide 5) Primary antibody raised against native protein but sample was denatured 6) Antibody concentration far below working range Solutions: 1) Verify primary antibody targets same species sequence as sample using NCBI protein database; confirm cross-reactivity in datasheet 2) Use secondary antibody raised in different species than primary antibody and phylogenetically distant from sample species 3) Match antibody classes: verify primary antibody class (usually IgG) and select corresponding secondary antibody 4) Remove all sodium azide from buffers when using HRP detection 5) Run non-denaturing gel or select antibody validated against denatured antigen 6) Increase antibody concentration 10-100 fold; use Luminata chemiluminescent reagents for higher sensitivity detection