Fluorescent Multiplex Detection of Proteins in Western Blots

Bio-Rad Laboratories Inc. has launched StarBright™ Blue 700, a fluorescent dye labeled secondary antibody for use in multiplex western blotting with a two- to fourfold lower limit of detection than IR800 labeled antibodies, the current industry standard. The company is also launching hFAB™ Rhodamine Anti-Housekeeping Protein Primary Antibodies for one-step detection of housekeeping proteins using western blots.

Detecting and quantifying multiple proteins simultaneously using western blotting can be challenging. Researchers are faced with performing either several time-consuming and error-prone chemiluminescent western blots or one fluorescent blot, which involves waiting up to 10 minutes to detect a signal that might be obscured by high background noise due to nonspecific binding.

To address those workflow challenges, Bio-Rad created StarBright Blue 700, a line of secondary antibodies that offer highly sensitive fluorescent detection, short exposure times, and easy multiplexing for western blotting.

The StarBright Blue 700 Secondary Antibodies are labeled with a fluorescent dye that is exceptionally bright thanks to the multiple donor-acceptor pairs present in each polymer molecule that allow it to efficiently accept and emit light. The 700 nm (near infrared) emission of StarBright Antibodies results in minimal background signal, and the conjugation to highly cross-adsorbed IgG leads to low nonspecific binding.

The StarBright Blue 700 Secondary Antibodies have exposure times that are 30 to 100 times shorter than IR800 labeled antibodies. Instead of 5 to 10 minutes, StarBright Antibodies need only a few seconds of light exposure to detect even the least abundant protein of interest, thus reducing potential background noise.

“Because of their high sensitivity of detection and ease of use with other antibodies for multiplexing, including stain-free and/or other IR800 labeled antibodies, StarBright Secondary Antibodies allow researchers to easily identify and quantify up to three unique proteins on one blot,” said Poulomi Acharya, PhD, Bio-Rad Global Product Manager, Protein Quantitation.

One-step detection of housekeeping proteins

For scientists who want to continue using their validated housekeeping proteins for protein normalization, Bio-Rad also offers human fragment antigen-binding (hFab) primary antibodies prelabeled with rhodamine dye. The antibodies are generated using Bio-Rad’s Human Combinatorial Antibody Library (HuCAL®) technology and can be used with other primary/secondary antibodies from different species without cross-reactivity. They are versatile, detecting human, rat, and mouse housekeeping proteins (actin, tubulin, GAPDH).

These labeled primary antibodies offer convenient one-step detection of common housekeeping proteins without the need for a secondary antibody, making them more reliable and easier to use than traditional anti-housekeeping antibodies.

Both products are part of Bio-Rad’s family of instruments, reagents, and antibodies designed to obtain fluorescent western blot results reliably, reproducibly, and with high-quality data. With the recently launched ChemiDoc™ MP Imaging System, a highly sensitive and flexible fluorescent detection imaging platform, and a portfolio of unique fluorescent-labeled secondary and primary antibodies, Bio-Rad is one of the leading providers of end-to-end western blotting workflow solutions.

For researchers new to western blot imaging, Bio-Rad continues to lead the industry in customer support and online resources, including Western Blot Doctor™ and a well-stocked library of videos.