ultra-high-speed-western-blot-using-immunoreaction-enhancing

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and ...

National Institutes of Health

Gifu University

The immunoblotting technique (also known as western blotting) is an essential tool used in biomedical research to determine the relative size and abundance of specific proteins and protein modifications. However, long incubation times severely limit its throughput. We have devised a system that improves antigen binding by cyclic draining and replenishing (CDR) of the antibody solution in conjunction with an immunoreaction enhancing agent. Biochemical analyses revealed that the CDR method reduced the incubation time of the antibodies, and the presence of a commercial immunoreaction enhancing agent altered the affinity of the antibody, respectively. Combination of the CDR method with the immunoreaction enhancing agent considerably enhanced the output signal and further reduced the incubation time of the antibodies. The resulting high-speed immunoblot can be completed in 20 min without any loss in sensitivity. Further, the antibodies are fully reusable. This method is effective for both chemiluminescence and fluorescence detection. Widespread adoption of this technique could dramatically boost efficiency and productivity across the life sciences.

Old but not obsolete: an enhanced high-speed immunoblot.

Complex glycans play vital roles in many biological processes, ranging from intracellular signaling and organ development to tumor growth. Glycan expression is routinely assessed by the application of glycan-specific antibodies to cells and tissues. However, glycan-specific antibodies quite often show a large number of bands on immunoblots and it is hard to interpret the data when reliable controls are lacking. This limits the scope of glycobiology studies and poses challenges for replication. We sought to resolve this issue by developing a novel strategy that utilizes an immunoreaction enhancing technology to vastly improve the speed and quality of glycan-based immunoblots. As a representative case study, we used chondroitin sulfate glycosaminoglycan (CS-GAG) chains as the carbohydrate target and a monoclonal antibody, CS-56, as the probe. We discovered that pre-incubation of the antibody with its antigenic CS-GAG chain distinguishes true-positive signals from false-positive ones. We successfully applied this strategy to 10E4, a monoclonal anti heparan sulfate GAGs (HS-GAGs) antibody, where true-positive signals were confirmed by chemical HS-GAG depolymerization on the membrane. This evidence that glycan-specific antibodies can generate clear and convincing data on immunoblot with highly replicable results opens new opportunities for many facets of life science research in glycobiology.

Haruna Nagase

Laboratory of Developmental Neurobiology,
Cell and Developmental Biology Center,
National Heart,
Lung,
and Blood Institute,
Laboratory of Developmental Neurobiology, Cell and Developmental Biology Center, National Heart, Lung, and Blood Institute

National Institutes of Health

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology