Cell lysates prepared from 3T3-L1 cells were separated by SDS-PAGE, transferred to PVDF membranes and incubated with peroxidase-conjugated avidin. Peroxidase-conjugated avidin interacted with bands of proteins with estimated molecular weights of 120, 74, and 72 kDa. This interaction was not limited to 3T3-L1 cells, because peroxidase-avidin also interacted with these 3 proteins in MC3T3-E1, YROS-1, Saos-2, MG63, SCCKN, and SCCTF cells although the staining intensity was different in each cell type. Cell lysates prepared from 3T3-L1 cells were also analyzed by avidin-biotin complex Western blotting method using the anti-Bax antibody. The antibody interacted with bands of proteins with estimated molecular weights of 120, 74, 72, and 25 kDa. However, only the 25-kDa band was detected with the anti-Bax antibody when the direct immunoblotting method was used. Peroxidase-conjugated avidin interacted with these 3 proteins in the mitochondria-containing fractions prepared from 3T3-L1 cells. However, these proteins were not detected in the fractions containing nucleus or peroxisome. FITC-avidin was also localized in mitochondria in the cultured cells. The localization of avidin-interacting proteins in mitochondria was confirmed by using rhodamine-avidin or by double staining with FITC-avidin and Mito-tracker. When attempting to use avidin-biotin complex immunoblotting and immunohistochemical procedures to detect the specific proteins in cultured cells, the avidin-interacting proteins demonstrated in the present study will be detected and will give non-specific staining if the corresponding concentrations of peroxidaseconjugated avidin are used. The present study also indicates that careful observation should be taken when using the avidin-biotin complex system in biochemical and histochemical examination
