Speckled or Spotted Background Pattern (Western Blot Immunodetection)

Question

Accepted Answer

Discrete spots or speckles appear across the membrane background rather than uniform signal. Pattern resembles aggregates or particulates. Common causes: 1) Protein aggregates in blocking reagent solution (BSA, milk powder) 2) HRP-conjugated secondary antibody contains aggregates 3) Insufficient washing after antibody incubations 4) Particulates in reagent solutions Solutions: 1) Filter blocking reagent through 0.2 µm or 0.45 µm Millex syringe filter before use 2) Filter HRP-conjugated secondary antibody solution through 0.2 µm or 0.45 µm Millex syringe filter 3) Increase number and duration of wash steps after primary and secondary antibody incubations 4) Pre-filter all solutions including transfer buffer through 0.2-0.45 µm filters