Fluorescent Western blotting

Fluorescent Western blotting allows multiplex detection without stripping and reprobing for analytes, and can be used for quantitative detection. Various fluorescent conjugates can be employed depending on the instrument setup. For optimal detection, Alexa Fluor® 680 and Alexa Fluor® 790 conjugates can be used to achieve high sensitivity Western blots.

Read more about the fluorescent conjugates offered by Jackson ImmunoResearch

Near-Infrared (NIR) Fluorescent Conjugates

Antibodies conjugated with far-red- and infrared-emitting dyes are suitable for a variety of techniques requiring the highest sensitivity. Alexa Fluor® 680 and 790 dyes are more sensitive than visible light-emitting dyes due to lower fluorescence quenching of the conjugates and higher extinction coefficients of the dyes. Low sample autofluorescence in this region of the spectrum results in lower background signal compared with other fluorophores.

Alexa Fluor® 680 and 790 conjugates can be used for high sensitivity Western blots, quantitative Western blots, in-gel Western blots, microWestern arrays, in-cell Western assays, on-cell Western assays, and other techniques that require the brightest dyes.

Multiplex detection of multiple protein targets without stripping and reprobing, using Alexa Fluor® 680 far-red dye and Alexa Fluor® 790 infrared dye. Mouse IgG was reduced and denatured with β‑mercaptoethanol and SDS. The heavy and light chains were separated by SDS-PAGE, transferred to nitrocellulose, and double labeled with a 1:100,000 dilution of Alexa Fluor® 790-Goat Anti‑Mouse IgG, FcY Subclass 1 specific (min X Hu, Bov, Rb Sr Prot, 115-655-205) (green) detecting heavy chains at 50 kDa and a 1:100,000 dilution of Alexa Fluor® 680-Goat Anti‑Mouse IgG, light chain specific (min X Bov, Gt, Hrs, Hu, Rb, Rat, Shp Ig, 115-625-174) (red) detecting light chains at 25 kDa). Fluorescence was imaged in a LI-COR Odyssey® imager.

Other Applications

The increased brightness of far-red and infrared dye conjugates (Alexa Fluor® 680 and 790) allows for a wide range of immunofluorescence detection and imaging modalities.

Fluorescence microscopy

Alexa Fluor® 680 and 790 secondary antibodies are adsorbed to minimize cross-reactions with others species and/or with other immunoglobulin classes, facilitating multiple labeling.

Flow cytometry

With modern flow cytometers now able to accommodate longer wavelengths, Alexa Fluor® 680 and 790 provide expanded choice for flow cytometry dye panels.

Format

Jackson ImmunoResearch offers a comprehensive selection of Alexa Fluor® 680 and Alexa Fluor® 790 dyes conjugated to secondary antibodies, streptavidin, signal enhancing antibodies and purified Donkey, Goat and Mouse IgG controls.