Blocking Buffer For Fluorescent Western Blotting

Blocking buffer for Western Blotting was prepared using ultra pure reagents dissolved in pharmaceutical grade water (WFI) and consists of a proprietary protein formulation in TRIS buffered saline at pH 7.6 with thimerosal added as an antimicrobial agent.

Fluorescence technology is widely used to detect proteins. However, many common visible fluorophores often result in considerable background fluorescence in the visible range. Visible fluorophores are rarely used for membrane-based protein detection because of this high background. IRDye800, IRDye700DX, Alexa Fluor 680 and Cy5.5 antibody and reagent conjugates are specifically designed for protein detection methods that use longer-wavelength, near-infrared (IR) fluorophores to visualize proteins in western blotting and other applications. IRDye800 and IRDye700DX fluoresce outside the range of most autofluorescence and therefore specific signal is sharply evident from any background giving the best possible signal-to-noise ratio. Detection levels in the picogram range on Western blots rival the sensitivity of chemiluminescence on film. IRDye800 conjugates are also suitable for immunofluorescence microscopy using commercially available excitation/emission filters in the 780nm/820nm range. Dual simultaneous labeling in western blots or microscopy is achieved when IRDye800 conjugates are used in conjunction with IRDye700 or Cy5.5 conjugates. IRDye800 and IRDye700DX conjugates provide an ultra-sensitive and convenient alternative to standard chemiluminescent protein detection methods, as well as a valuable tool for multicolor imaging. Once reacted with the membrane and dried, IRDye800 and IRDye700DX conjugated antibody-protein complexes are very stable, and membranes can be stored protected from light, re-washed and/or rescanned. This blocking buffer is specifically formulated to achieve superior reproducible western blotting images using this system.