The main causes of high background in WB experiments include excessive primary antibody concentration and overlong incubation time, insufficient washing cycles or inadequate rinsing; insufficient blocking duration or low concentration of BSA/skim milk in blocking buffer; over-transfer leading to excessive protein adsorption on the membrane; non-specific binding of secondary antibodies; prolonged film exposure time; residual miscellaneous proteins in gels, and contaminated experimental consumables can also result in overall high background.