The effect of prestaining prior to immunoreaction during electrophoretic blotting of proteins

Abstract

AbstractWe have used six different monoclonal and polyclonal antibodies, four different antigen preparations and two different detection systems to compare Western blotting with Coomassie Blue prestained gels (Jackson and Thompson, Electrophoresis, 1984, 5, 35–42) to blotting of unstained gels with Amido Black or Fast Green post‐transfer staining of the nitrocellulose. Contrary to a recent report (Harper et al., Anal. Biochem., 1986, 157, 270–274), in which post‐staining with Coomassie Blue was determined to severely inhibit immunoreactivity, we find using prestained Coomassie Blue gels to be extremely useful in most cases, although, rarely, inhibition of the immune reaction may take place. Post‐staining with Amido Black or Fast Green may also prove useful, but is not recommended since similar inhibition occurs, and there are the added disadvantages of not being able to “pre‐view” the gel pattern and not being able to use stored gels. Further studies indicated that the rare loss of immunoreactivity seen with Coomassie Blue prestained gels is most likely due to the necessary fixation step, and not the stain itself (as has been suggested), since the protein‐dye complex is dissociated during transfer.