Figure 2.

Effect of blocking buffer on selectivity of an anti-cofilin primary antibody. Cofilin (∼19 kDa) was detected on blots blocked with 5% BSA, 5% nonfat dry milk, or Odyssey blocking buffer. Blots were visualized with IRDye 800CW secondary antibody and laser-based digital imaging. All blots were processed identically and imaged together, with blocking buffer as the only variation. Tissue lysates are as follows: lane 1, mouse brain; lane 2, rat brain; lane 3, mouse liver; lane 4, rat liver; lane 5, mouse thymus; and lane 6, rat thymus. Arrow indicates the expected position of the 19-kDa cofilin band. Choice of blocking buffer greatly impacted the off-target binding of this antibody. For all primary antibodies tested in this study, BSA consistently produced more nonspecific bands than the other blocking buffers. Reprinted with permission from Ambroz et al. (62).