HiGlo Blocking Buffer for Fluorescent Western Blotting

HiGlo Blocking Buffer is specifically designed for western blotting using fluorochrome conjugated antibodies.

Pure nitrocellulose membrane is recommended for maximum performance. Other membranes, such as PVDF or nitrocellulose embedded in a support can be used, but may generate elevated backgrounds. Protein should be transferred from gel to membrane using standard protocols. This buffer can be used for membrane blocking and to dilute both primary and secondary antibodies. The buffer is suitable for use with fluorescent western blot imaging systems produced by Bio-Rad Laboratories, GE Healthcare, Alpha Innotech, FujiFilm Life Science, Licor Biosciences, UVP, Syngene and The BlotCycler™ from Precision Biosystems.

Fluorescence technology is widely used to detect proteins in both the visible and near-infrared ranges. HiGlo blocking buffer allows for superior signal detection and lower background noise when fluorochrome conjugated antibodies are used to visualize proteins in western blotting and other applications. Antibody conjugates prepared with IRDye® 800 and IRDye® 700DX (Licor), Cy2™, Cy3™, Cy3.5™, Cy5™ and Cy5.5™ (GE Healthcare), DyLight™405, DyLight™ 549, DyLight™ 649, DyLight™ 680, and DyLight™ 800 (Thermo Fisher/Pierce) and Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 647 and Alexa Fluor® 680 (Invitrogen/Molecular Probes) have been validated on various platforms using this product with superior results compared to other commercially available products. In the infrared range, where little to no autofluorescence occurs, specific signal is sharply evident from any background giving the best possible signal-to-noise ratio. This allows for detection levels in the picogram range which rivals the sensitivity of chemiluminescence on film for western blotting.

This product is also suitable for simultaneous labeling (multiplex) in western blots or microscopy using various fluorochrome combinations for multicolor imaging. Membranes blocked with this product can be dried and are very stable. Membranes that are stored protected from light can be re-washed and/or rescanned.

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