Enhanced Multiplex Western Blotting



Rockland Immunochemicals and Bio-Rad Laboratories have jointly developed an enhanced multiplex fluorescent western blotting protocol for both the production of an immunoblot on PVDF membrane and determining the limit of fluorescent detection. The membrane is probed with specific primary antibodies and DyLight™ fluorochrome conjugated secondary antibodies developed by Rockland Immunochemicals.

Optimized detection is achieved using the Molecular Imager® VersaDoc™ MP 4000 imaging system with additional analysis facilitated by using Quantity One® 1-D software developed by Bio-Rad Laboratories. We highlight two examples of the simultaneous detection of several human serum proteins, and the simultaneous detection of resting Akt1 and activated phosphorylated Akt1. Each blot was reacted with specific primary antibodies reactive with target proteins. The blots were probed simultaneously with two or three different DyLight™ conjugated secondary antibodies specific for primary antibodies each raised in a unique host animal (mouse, rabbit, and goat). The resulting blots show enhanced detection of distinct target bands labeled by a unique identifying color.