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Multiplexed Westerns without Compromises

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Wondering how to get more data from your western blots? The answer is fluorescent multiplex western blotting. Multiplexing saves valuable sample and time and eliminates the need to strip, reprobe, or cut your blot. We are here to help you transition to the next level of western blotting with videos, protocols, tips, and more. Read on to explore Bio-Rad’s complete solution for fluorescent western blotting.


Why Do Fluorescent Westerns?



Recent advances in digital imaging technology and fluorescent western blotting reagents have resulted in better sensitivity and data quality.


Advantages of fluorescent western blotting:

  • More data
  • Easier multiplexing
  • Less sample used
  • Fewer reagents used
  • No stripping, reprobing, or blot cutting
  • Easier method development
Chemiluminescence

Chemiluminescence

1 Color

Traditional Fluorescence

Traditional Fluorescence

2 Colors

Bio-Rad Fluorescence

Bio-Rad Fluorescence

3 Colors + Total Protein Normalization

Learn more about our Fluorescent Western Blotting System


How to Do Fluorescent Westerns?



Fluorescent western blotting doesn't have to be difficult. Here are a few resources to get you started.

Western Blotting Fluorophores — Excitation and Emission Poster

Use this poster to:

  • Reference the excitation and emission spectra of commonly used fluorophores
  • Choose the right fluorophores to run multiplex experiments
  • Brighten up your lab
Request a Free Copy

Handy Tips for Fluorescent Western Blotting

Hover over each image for more information.

High background

Seeing high fluorescence background? Try specialized fluorescence blocking buffers, like those with casein. Also consider using LF-PVDF membranes.

Blown out standards

Standards too bright? Avoid using standards that contain pink bands as these fluoresce very readily. Try Precision Plus Protein™ All Blue or Precision Plus Protein Unstained Standards instead.

Pen and other artifacts

Don't want to see all the marks on your membrane? Mark the membrane with a pencil, not an ink pen.

Want a demo of the new fluorescent western blotting imaging system?


See Products and Resources



Fluorescent Western Blotting Workflow

Bio-Rad's fluorescent western blotting workflow is a seamless integration of products designed to work together to offer guaranteed results. This validated set of solutions will make it easy for you to get better data every time.

Request a demo of the entire V3 Western Workflow

Introduction

Better Options

Better Data

Better Experience

Move beyond just traditional imaging

Move beyond traditional laser scanning systems and X-ray film. Get the fluorescence performance you expect with higher multiplexing (three targets vs. two) and more fluorophore options. The performance of film meets the quantitation capabilities of digital.

This is the ChemiDoc™ MP Imaging System.

Fluorescent Western Blotting
ChemiDoc MP Imaging System

Why compromise when you can get the best of chemiluminescence and fluorescence in a single imaging system.

 

Traditional systems offer limited options.

10 sec

Traditional Film

Film

OR

200 sec

Traditional Fluorescence Scanner

Fluorescence Scanner

With the ChemiDoc MP System, the superior quantitation of digital meets the sensitivity of
film and delivers the best fluorescence performance with the brightest new fluorophore.

10 sec

ChemiDoc MP System Chemiluminescence

Chemiluminescence

AND

200 sec

ChemiDoc MP System IR800 Fluorescence

IR800 Fluorescence

AND

20 sec

ChemiDoc MP System StarBright Blue 700 Fluorophore

StarBright™ Blue 700 Fluorophore

See up to three signals at once or look at a subset of signals. Plus, you can normalize beyond a single protein with stain-free total protein normalization.


Please deselect stain-free to
explore blot combinations.

Stain-Free Total Protein Signal Phospho-p44/42 MAPK (ERK1/2) — StarBright B700 Total p44/42 MAPK (ERK1/2) — DyLight 800 GAPDH — hFAB Rhodamine Phospho-p44/42 MAPK (ERK1/2) — StarBright B700, Total p44/42 MAPK (ERK1/2) — DyLight 800 Phospho-p44/42 MAPK (ERK1/2) — StarBright B700, GAPDH — hFAB Rhodamine Total p44/42 MAPK (ERK1/2) — DyLight 800, GAPDH — hFAB Rhodamine Phospho-p44/42 MAPK (ERK1/2) — StarBright B700, Total p44/42 MAPK (ERK1/2) — DyLight 800, GAPDH — hFAB Rhodamine

Explore blot combinations by making a selection on the right.

Select Blots Below:









The stain-free channel is used in addition to the three other channels, but cannot be used at the same time. Learn more about stain-free here.

Image Lab Touch Software:

  Teaches as you tap and explore           Picks optimal light source and filters           Suggests exposure times

Image Lab Touch Software

Want to learn more?


Talk to a Specialist

Need to see to believe?

Bio-Rad presents the StarBright™ Blue 700 Secondary Antibodies, which are fluorescently tagged detection reagents for western blotting. These tagged antibodies are exceptionally bright and stable to photobleaching and offer unparalleled sensitivity and reproducibility in western blotting.

Bio-Rad's hFAB™ Rhodamine Antibodies are fluorescently labeled primary antibodies raised against the housekeeping proteins (HKPs) actin, tubulin, and GAPDH. They are intended to be used for normalizing protein loading in western blotting experiments. The hFAB Rhodamine Antibodies require no secondary antibodies to detect HKPs.

470 nm

700 nm

StarBright Blue 700 Secondary Antibodies

Excitation

Energy
transfer

Emission

Light-absorbing
monomer

Energy transfer
monomer

Light-emitting
monomer

Linker monomer for
functionalization

Key Advantages

Easy Multiplexing

StarBright Secondary Antibodies can be used with IR800 antibodies or other common fluorophores to detect multiple proteins on the same blot. Bio-Rad's stain-free technology and/or the hFAB Rhodamine Anti-Housekeeping Antibodies can be used to normalize protein loading.

High Signal, Minimum Noise

The StarBright fluorophore (maximum excitation/
emission = 470 nm/700 nm) emits in the far red/near infrared (NIR) range of the spectrum, where there is minimal background.

Shorter Exposure Time

The exceptional brightness of StarBright Antibodies leads to exposure times orders of magnitude shorter than those of regular fluorescent dyes.

Low Nonspecific Binding

The StarBright fluorophore is conjugated to highly cross-adsorbed antibodies.

See StarBright and hFAB Antibodies brochures and documents ▸

Bio-Rad's hFAB Rhodamine Antibodies are fluorescently labeled primary antibodies raised against the actin, tubulin, and GAPDH housekeeping proteins (HKPs).

  • Intended for normalizing protein loading in western blotting experiments
  • Sufficient to detect HKPs; secondary antibodies are not needed for detection
hFAB Rhodamine Antibodies

Browse and Purchase Fluorescent Western Blotting Products

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