Samples are first separated by SDS-polyacrylamide gel electrophoresis. These membranes are incubated with an antibody specific for the protein of interest which binds to the protein band immobilized on the membrane. The antibody is then visualized with a detection system that is usually based on a secondary protein binding to Ig chains which is linked to a color-yielding reaction. It is not uncommon that, contrary to the theoretical predictions, several bands are detected. This is not uncommon, particularly if samples are stored for prolonged time or if proteins or membranes are fractionated after homogenization of the starting tissue.
All additional bands are of lower apparent molecular mass than the full-length protein.
Particularly susceptible are synapsins and synaptotagmins. Addition of protease inhibitors such as PMSF, pepstatin or leupeptin should be considered. Too much protein per lane or detection system too sensitive . Immobilized proteins may provide a concentrated adsorbtive surface to which certain IgG may bind nonspecifically. Similarly, such nonspecific binding may be uncovered when highly sensitive detection systems such as enhanced chemoluminescence are employed.