Multiplex Fluorescent Western Blotting

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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:

• Multiple samples are analyzed under the same experimental conditions
• Results in context by simultaneous analysis of several proteins and parameters
• Greater consistency of readout using fluorescent dyes rather than the variability of an enzymatic reaction
• Amount of information collected is maximized, using as little of your rare/precious sample as possible
• Cost and time savings

Bio-Rad offers a new generation of multiplex western blotting reagents and equipment, that includes:

• Fluorescent StarBright™ Blue 700 and 520 Secondary Antibodies
• hFAB™ Rhodamine Anti-Housekeeping Protein Primary Antibodies
• Imaging with ChemiDoc™ MP Imaging System

Read the introductory guide for detailed information on multiplex fluorescent western blotting.

StarBright Blue Secondary Antibodies

StarBright Blue Secondary Antibodies are labeled with exceptionally bright and stable semi-conducting 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.

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.

Advantages of StarBright Blue Secondary Antibodies compared to other traditional fluorophores:

• Highly sensitive fluorescent detection — 2- to 4-fold lower limit of detection than traditional fluorophores
• Short exposure times — 50- to 100-fold shorter (seconds vs. minutes)
• Easy multiplexing options — compatible with stain-free gel and blot imaging detection and hFAB Rhodamine Antibodies as well as antibodies labeled with traditional fluorophores

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.

StarBright Blue 700 Secondary Antibody Range

# Catalog	Description
12004158	Goat Anti-Mouse IgG StarBright Blue 700, 400 µl
12004159	Goat Anti-Mouse IgG StarBright Blue 700, 80 µl
12004161	Goat Anti-Rabbit IgG StarBright Blue 700, 400 µl
12004162	Goat Anti-Rabbit IgG StarBright Blue 700, 80 µl

StarBright Blue 520 Secondary Antibody Range

# Catalog	Description
12005866	Goat Anti-Mouse IgG StarBright Blue 520, 400 µl
12005867	Goat Anti-Mouse IgG StarBright Blue 520, 80 µl
12005869	Goat Anti-Rabbit IgG StarBright Blue 520 400 µl
12005870	Goat Anti-Rabbit IgG StarBright Blue 520, 80 µl

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hFAB Rhodamine Housekeeping Protein Fluorescent Primary Antibodies

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.

hFAB Rhodamine Antibody Range

# Catalog	Description
12004163	Anti-Actin hFAB Rhodamine Antibody, 200 µl
12004164	Anti-Actin hFAB Rhodamine Antibody, 40 µl
12004165	Anti-Tubulin hFAB Rhodamine Antibody, 200 µl
12004166	Anti-Tubulin hFAB Rhodamine Antibody, 40 µl
12004167	Anti-GAPDH hFAB Rhodamine Antibody, 200 µl
12004168	Anti-GAPDH hFAB Rhodamine Antibody, 40 µl

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Imaging with ChemiDoc MP Imaging System and Image Lab Touch Software

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
 

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:

• Sensitive detection with low fluorescence background
• Chemiluminescence sensitivity matching X-ray film
• Easy-to-use software
• Multiplex fluorescence detection

Learn more about the imaging system and software, including full product specification, user guides and resources.

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Products for Western Blotting

PrecisionAb Western Blotting Primary Antibodies

Reproducibility at your fingertips.

Fluorescent Western Botting Antibodies

Multiplex western blotting made easy.

ChemiDoc MP Imaging System

Fluorescence and chemiluminescence detection without compromises.

Clarity and Clarity Max Western ECL Blotting Substrates

Clarity family of ECL substrates provides high performance for all your western chemiluminescence needs.