Fluorescent immunoblot imaging
The increasing need to get multi-parameter measurements 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 thereby obtaining as much information at one time. The benefits of fluorescent western blotting multiplexing are;
Bio-Rad brings to you a new generation of multiplex western blotting reagents and equipment, that includes;
Read the introductory guide that will provide detailed information on this multiplex fluorescent western blotting.
StarBright Blue 700 Secondary Antibodies are labeled with exceptionally bright and stable semiconducting polymer nanoparticles. The maximal excitation occurs with blue light (470 nm) and emission is restricted to a narrow far red/near infrared region of the spectrum (700 nm). These unique properties make StarBright Blue 700 Secondary Antibodies ideal for multiplex western blotting. Figure 1 shows a composite blot image with three distinct proteins visualized using the StarBright Blue 700 Secondary Antibody in combination with a rhodamine labeled recombinant human hFAB antibody for the detection of popular housekeeping proteins on the ChemiDoc MP Imaging System.
Fig. 1. Composite blot image for triplex western blotting.
Rabbit Anti-Human ATG7 PrecisionAb™ Antibody (78 kD)
Goat Anti-Rabbit IgG StarBright Blue 700 Antibody(12004161)
Mouse Anti-Human AKR1C2 PrecisionAb Antibody (36 kD)
Goat Anti-Mouse IgG DyLight 800 Conjugation Kit (STAR117D800GA)
Rhodamine Antibody (50 kD)
PrecisionAb Antibodies were chosen for this triplex western blot because of their superior performance and reproducibility, having been rigorously tested and selected for their 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 700 Antibodies instruction manual and the Fluorescent Western Blotting Antibodies - Experience Unmatched Sensitivity brochure.
These hFAB antibodies are rhodamine fluorescently labeled primary antibodies recognizing the major housekeeping proteins (HKPs) actin, tubulin and GAPDH. Although these antibodies have been raised against human immunogens they are compatible for the detection of housekeeping proteins due to the fact that these proteins are evolutionary conserved, making them ideal for the design of multiplexing experiments with mouse and rabbit antibodies. They are intended for normalizing protein loading in western blot 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 700 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.
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.
Visualization of proteins in SDS-PAGE gels without the need for staining or destaining of gels. The trihalo compounds within the gel are covalently linked to tryptophan residues in proteins when activated by UV light. Activation results in addition of a 58 Dalton moiety to each tryptophan, which fluoresces after another brief exposure to UV light, enabling protein visualization.
Stain free technology therefore allows the normalization of western blot band intensities against total protein levels from the sample loaded onto the gel. This enables you to perform subsequent relative quantification analysis. Mini and midi sized stain free precast gels, which contain trihalo compounds, are available. View an example of a stain free technology in an IP experiment using TidyBlotTM 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.
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