Bio-Rad Laboratories has introduced StarBright Blue 520 Fluorescent Secondary Antibodies, fluorescent dye–labelled secondary antibodies for use in multiplex western blotting. They exhibit a two- to threefold lower limit of detection than traditional green emission fluorophore-labelled antibodies such as Alexa Fluor 488 or DyLight 488, the current industry standards.
Using traditional chemiluminescent methods to detect and quantify multiple proteins simultaneously can be challenging. Scientists often resort to cutting or reprobing blots, which introduces variability and results in protein loss, thereby rendering the blot irreproducible and unsuitable for quantitation. An alternate method involves attempting to sequentially probe a single blot with multiple fluorescent antibodies to distinguish the signals from each one; however, these protocols are cumbersome to optimise and execute.
To address these workflow challenges, Bio-Rad created the StarBright Blue line of secondary antibodies that enable highly sensitive fluorescent detection, short exposure times, and easy multiplexing for western blotting. These plug-and-play antibodies allow simultaneous detection of up to three proteins (two targets of interest and one housekeeping protein) on the same blot when used with the hFAB Rhodamine Housekeeping Protein Fluorescent Primary Antibodies. The antibodies work seamlessly on nitrocellulose or low-fluorescence PVDF membranes and all other aspects of the standard western blotting workflow remain unchanged.
The StarBright Blue 520 Fluorescent Secondary Antibodies are labelled with a particularly bright fluorescent dye, resulting in short exposure times and a high signal-to-noise ratio. This property stems from the presence of multiple donor-acceptor pairs in each polymer molecule, which enable it to efficiently absorb and emit light. The product’s 520nm emission wavelength means the StarBright Blue 520 Fluorescent Secondary Antibody can pair with a StarBright Blue 700 Fluorescent Secondary Antibody or with other traditional fluorescent antibodies. It conjugates to highly cross-adsorbed IgG, which leads to low nonspecific binding.
