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Fluorescent immunoblot imaging
The increasing need to maximize data from a single sample has led to the development of fluorescent based multiplexing assays. Multiplex fluorescent western blotting enables the simultaneous analysis of multiple proteins in a single sample on the same blot, obtaining as much information as possible in one experiment.
The benefits of fluorescent western blotting multiplexing are:
Bio-Rad offers a new generation of multiplex western blotting reagents and equipment, that includes:
Read the introductory guide for detailed information on multiplex fluorescent western blotting.
StarBright Blue Secondary Antibodies are labeled with exceptionally bright and stable semi-conducting polymer nanoparticles. Their fluorescence emission is restricted to a narrow spectral band, making them ideal for multiplex fluorescence imaging for western blotting. StarBright Secondary Antibodies are excited with blue light (440-470 nm) and are available in two colors: StarBright Blue 520 emits in the green range of the spectrum (emission maximum: 520 nm) and StarBright Blue 700 maximally emits in the far red/near infrared region of the spectrum (700 nm). They can be used together or with other infrared or visible light emitting dye-conjugated antibodies for multiplex imaging applications.
Fig. 1. Composite blot image for quadruple western blotting.
Rabbit Anti-Human ATG7
Goat Anti-Rabbit IgG StarBright Blue 700
Mouse Anti-Human AKR1C2
Goat Anti-Mouse IgG StarBright Blue 520
Goat Anti-Human TP1 (biotinylated)
DyLight 800 Streptavidin
Figure 1 shows a composite blot image with four distinct proteins visualized using StarBright Blue 520 and StarBright Blue 700 Secondary Antibodies, in combination with Dylight 800 and a rhodamine labeled recombinant human hFAB antibody, for the detection of popular housekeeping proteins on the ChemiDoc MP Imaging System.
PrecisionAb Antibodies were chosen for this multiplex western blot because of their superior performance and reproducibility, having been rigorously tested and selected for high specificity, high sensitivity, and consistency.
Learn more about the validation process and customers’ experience using PrecisionAb Antibodies and the full range available.
Instructions for use, tips, troubleshooting and further information are available in the StarBright Blue Antibodies instruction manual and Comprehensive Guide to Fluorescence Western Blotting instruction manual.
Goat Anti-Mouse IgG StarBright Blue 700, 400 µl
Goat Anti-Mouse IgG StarBright Blue 700, 80 µl
Goat Anti-Rabbit IgG StarBright Blue 700, 400 µl
Goat Anti-Rabbit IgG StarBright Blue 700, 80 µl
Goat Anti-Mouse IgG StarBright Blue 520, 400 µl
Goat Anti-Mouse IgG StarBright Blue 520, 80 µl
Goat Anti-Rabbit IgG StarBright Blue 520 400 µl
Goat Anti-Rabbit IgG StarBright Blue 520, 80 µl
hFAB antibodies are primary antibodies directly labeled with rhodamine which recognize the major housekeeping proteins (HKPs) actin, tubulin and GAPDH. Although these antibodies have been raised against human immunogens, they are compatible for the design of multiplexing experiments with mouse and rabbit antibodies, as these housekeeping proteins are evolutionarily conserved. hFAB Rhodamine Housekeeping Proteins are intended for normalizing protein load in western blots without the need for a secondary antibody. The fluorescent properties (excitation 530 nm/emission 580 nm) of these antibodies make them highly compatible with StarBright Blue Antibodies and other secondary antibodies in multiplexing experiments.
Instructions for use, tips, troubleshooting and further information are available in the hFAB Rhodamine Antibodies instruction manual and the Fluorescent Western Blotting Antibodies - Experience Unmatched Sensitivity brochure.
Anti-Actin hFAB Rhodamine Antibody, 200 µl
Anti-Actin hFAB Rhodamine Antibody, 40 µl
Anti-Tubulin hFAB Rhodamine Antibody, 200 µl
Anti-Tubulin hFAB Rhodamine Antibody, 40 µl
Anti-GAPDH hFAB Rhodamine Antibody, 200 µl
Anti-GAPDH hFAB Rhodamine Antibody, 40 µl
ChemiDoc imagers offer visible light (RGB), far red/near infrared (FR/NIR) fluorescence and chemiluminescence detection. Stain-free imaging enables immediate visualization and verification of proteins without the need for gel staining. Image Lab Software is a family of software packages for the acquisition and analysis of gels and images.
Stain free technology allows the visualization of proteins in SDS-PAGE gels without the need for staining or destaining of gels. Trihalo compounds within the gel are covalently linked to tryptophan residues in proteins when activated by UV light. This allows the proteins to be visualized in gels or on PVDF membranes multiple times throughout the electrophoresis and blotting process for validation in a western blotting workflow.
Stain free technology therefore allows the normalization of western blot band intensities against total protein levels from the sample loaded onto the gel enabling you to perform subsequent relative quantification analysis. Mini and midi sized stain free precast gels, containing trihalo compounds, are available. View an example of a stain free technology in an IP experiment using TidyBlot Western Blot Detection Reagent.
Best in-class performance from the ChemiDoc MP Imaging System:
Learn more about the imaging system and software, including full product specification, user guides and resources.
Reproducibility at your fingertips.
Multiplex western blotting made easy.
Fluorescence and chemiluminescence detection without compromises.
Clarity family of ECL substrates provides high performance for all your western chemiluminescence needs.