western blotting

Multiplex Fluorescent Western Blotting

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;

  • Multiple samples 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 maximized while using as little of your rare/precious sample as possible
  • Cost and time saving

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

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.

Fig. 1. Composite blot image for triplex western blotting.

Target Protein

Primary Antibody

Secondary Antibody

#1: ATG7

Rabbit Anti-Human ATG7 PrecisionAb™ Antibody (78 kD)

(VPA00230)

Goat Anti-Rabbit IgG StarBright Blue 700 Antibody(12004161)

#2: AKR1C2

Mouse Anti-Human AKR1C2 PrecisionAb Antibody (36 kD)

(VPA00346)

Goat Anti-Mouse IgG DyLight 800 Conjugation Kit (STAR117D800GA)

Housekeeping

Anti-Tubulin hFAB

Rhodamine Antibody (50 kD)

(12004165)

Not required

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.
 

Advantages of StarBright Blue 700 Secondary Antibodies compared to other traditional fluorophores:

  • Highly sensitive fluorescent detection - 2- to 4-fold lower limit of detection
  • Short exposure times - 50- to 100-fold shorter (seconds vs. minutes)
  • Easy multiplexing options - stain-free gels and/or use with hFAB Rhodamine Antibodies and antibodies labeled with other traditional fluorophores

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.

Our StarBright Blue 700 Secondary Antibody Range

# Catalog Description
12004157 Goat Anti-Mouse IgG StarBright Blue 700, 5 X 400 µl
12004158 Goat Anti-Mouse IgG StarBright Blue 700, 400 µl
12004159 Goat Anti-Mouse IgG StarBright Blue 700, 80 µl
12004160 Goat Anti-Rabbit IgG StarBright Blue 700, 5 X 400 µl
12004161 Goat Anti-Rabbit IgG StarBright Blue 700, 400 µl
12004162 Goat Anti-Rabbit IgG StarBright Blue 700, 80 µl

hFAB Rhodamine Housekeeping Protein Fluorescent Primary Antibodies

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.

Our 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

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

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:

  • 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. 


NEW Products for Western Blotting

PrecisionAb™ Western Blotting Primary Antibodies

PrecisionAb™ Western Blotting Primary Antibodies

Reproducibility at your fingertips.

NEW Fluorescent Western Blotting Antibodies

NEW Fluorescent Western Blotting Antibodies

Multiplex Western Blotting made easy.

ChemiDoc™ MP Imaging System

ChemiDoc™ Touch 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.