Corporate Banner
Satellite Banner
Scientific Community
Become a Member | Sign in
Home>News>This Article

Producing Reliable Quantitative Western Blot Data

Published: Monday, June 17, 2013
Last Updated: Monday, June 17, 2013
Bookmark and Share
The reporting of fold changes in protein expression from western blots is often viewed with skepticism due to questions about the validity of the technique as a quantitative method.

In the May issue of Molecular Biotechnology, researchers at Bio-Rad Laboratories, Inc. reported a method for producing quantitative and reproducible western blot data that could establish the technique’s reputation for reliable quantitation.

Western blotting has been practiced for several decades and is performed routinely in labs to assess the presence of specific target proteins in complex samples. Although the scientific community has historically reported fold changes in protein expression by measuring the differential density of the target protein bands among the samples on western blots, the technique is generally considered to yield only semi- or nonquantitative data because of the methods, reagents, and instruments used to generate these data. This shortcoming led the authors to undertake the present study.

“In my visits to labs, researchers having been asking me how to more rigorously approach western blotting quantitation,” said Dr. Sean Taylor, lead author and a Bio-Rad field applications specialist. “We wrote this article to demonstrate how the application of a simple workflow coupled with the use of updated reagent and instrument technologies can ensure the production of quantifiable results from chemiluminescent western blots.”

Accurate normalization for loading differences is a solvable challenge 
To ensure accuracy in western blotting, loading controls are used to normalize for errors that can result from imprecise protein estimation, pipetting inaccuracy, or uneven protein transfer. The most common loading controls are housekeeping proteins. However, these proteins are generally highly expressed in samples and are frequently overloaded in a gel lane with the much lower abundance target protein, thus reducing their usefulness for normalization.

In these instances, total protein normalization using stain-free technology may be a suitable alternative. The study found that using stain-free imaging to quantify the total amount of proteins in each lane yielded results that closely matched the confirmed amounts, supporting the validity of this normalization method. This result was in direct contrast to quantification using the relative density of GAPDH, a common housekeeping protein, which did not correspond well to the actual amounts due to signal saturation at high sample loads.

Modern imaging technology has a greater dynamic range than film
The study also dispelled the widely held view that film should be the method of choice for detecting chemiluminescent western blots. Using a twofold dilution series of a protein lysate, the researchers reported that the CCD-based imaging system provided a linear dynamic range nearly an order of magnitude greater than film (0.04–2.5 ng vs. 0.04–0.31 ng) for the probed target protein. This permitted accurate quantification of the relative target band density between the gel lanes with the imaging system versus film for the identical blots.

“This paper demonstrates how to validate antibodies using an imaging system to ensure that individual samples are diluted and loaded within the quantitative linear dynamic range of each antibody to produce excellent densitometric data,” Taylor said. “It also shows how stain-free technology is a great alternative to traditional housekeeping proteins for normalization.”

Click below to read the Molecular Biotechnology paper describing how to reliably quantify proteins in your next western blot experiment.

Further Information
Access to this exclusive content is for Technology Networks Premium members only.

Join Technology Networks Premium for free access to:

  • Exclusive articles
  • Presentations from international conferences
  • Over 2,800+ scientific posters on ePosters
  • More than 4,000+ scientific videos on LabTube
  • 35 community eNewsletters

Sign In

Forgotten your details? Click Here
If you are not a member you can join here

*Please note: By logging into you agree to accept the use of cookies. To find out more about the cookies we use and how to delete them, see our privacy policy.

Related Content

Droplet Digital PCR Enables Measurement of Potential Cancer Survival Biomarker
Study paves the way for further research into the role of TIL quantification in immunotherapy and as a cancer survival predictor.
Tuesday, December 10, 2013
Bio-Rad and Myriad RBM Partner to Develop Immunoassay Kits
Partnership to commercialize multiplex biomarker kits for life science research.
Wednesday, August 01, 2012
Scientific News
Boosting Breast Cancer Treatment
To more efficiently treat breast cancer, scientists have been researching molecules that selectively bind to cancer cells and deliver a substance that can kill the tumor cells, for several years.
Research Finding Could Lead to Targeted Therapies for IBD
Findings published online in Cell Reports.
A Cellular Symphony Responsible for Autoimmune Disease
Broad Institute researchers have used a novel approach to increase our understanding of the immune system as a whole.
Genetic Basis of Fatal Flu Side Effect Discovered
A group of people with fatal H1N1 flu died after their viral infections triggered a deadly hyperinflammatory disorder in susceptible individuals with gene mutations linked to the overactive immune response, according to a recent study.
Developing Drug Resistance may be a Matter of Diversity for Tuberculosis
Researchers have probed the bacteria that causes tuberculosis, Mycobacterium tuberculosis, to learn more about how individual bacterial cells change and adapt while in the human body.
Surprising Trait Found in Anti-HIV Antibodies
Scientists at The Scripps Research Institute (TSRI) have new weapons in the fight against HIV.
Some Gut Microbes May Be Keystones of Health
University of Oregon scientists have found that strength in numbers doesn’t hold true for microbes in the intestines. A minority population of the right type might hold the key to regulating good health.
Essential Component of Antiviral Defense Identified
Infectious disease researchers at the University of Georgia have identified a signaling protein critical for host defense against influenza infection.
Single Vaccine for Chikungunya, Related Viruses May be Possible
What if a single vaccine could protect people from infection by many different viruses? That concept is a step closer to reality.
Is Allergy the Price We Pay for Our Immunity to Parasites?
New findings help demonstrate the evolutionary basis for allergy.

SELECTBIO Market Reports
Go to LabTube
Go to eposters
Access to the latest scientific news
Exclusive articles
Upload and share your posters on ePosters
Latest presentations and webinars
View a library of 1,800+ scientific and medical posters
2,800+ scientific and medical posters
A library of 2,500+ scientific videos on LabTube
4,000+ scientific videos