Multiplex Fluorescent Western Blotting

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

StarBright Blue 520 Secondary Antibody Range

hFAB Rhodamine Housekeeping Protein Fluorescent Primary Antibodies

hFAB antibodies are primary antibodies, directly labeled with green-excitable rhodamine, which recognize the major housekeeping proteins (HKPs) β-Tubulin, β-Actin, 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.

These Fab fragments have engineered advantages:

• Ensure product consistency and data reproduciblity
• Free-up your multiplexing species choices for your primary targets: being preconjugated, they do not require secondary detection, and are not recognized by conventional secondary detection reagents so they can be used alongside any secondary antibodies without interference
• Facilitate load normalization without the need for a secondary antibody: hFAB Rhodamine Antibodies give a linear response in the common loading range of 10–50 μg of cell lysate, enabling different quantities (low and high) to be easily distinguished
• Are optimized for multiplexing (fluorescent properties: excitation 530 nm/emission 580 nm):
  • 3-color multiplexing with no crosstalk, when using StarBright Dyes for western blotting; the green fluorescence is intermediate between the blue fluorescence of StarBright 700 and StarBright 520
  • 5-color multiplexing in combination with DyLight 800 and Alexa 647 conjugated antibodies (Figure 2)

Fig. 2. Western blot of HeLa cell lysate probed with five different antibodies.

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

Imaging with ChemiDoc MP Imaging System and Image Lab Touch Software

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.