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

Avoiding Common Pitfalls of Quantitative Western Blot Experiments

Published: Friday, April 04, 2014
Last Updated: Friday, April 04, 2014
Bookmark and Share
Although a well-established laboratory technique, western blotting has recently come under fire as a quantitative method.

A new methods article provides a rigorous and concise workflow with specific instructions on how to produce and analyze quantitative data using western blot experiments. The paper, coauthored by Bio-Rad scientists and published in BioMed Research International, also highlights recently introduced technologies that improve reproducibility. The result is a powerful, step-by-step guide to obtaining quantitative and reproducible densitometric data from western blots regardless of the specific experiment.

The technique is challenging and requires following a rigorous methodology to achieve reproducible and quantitative data. According to a recent survey of more than 750 labs, 41% of researchers say their western blots fail a quarter of the time.

Dr. Aldrin Gomes, an assistant professor at University of California, Davis, agrees that flawed western blots are not unusual. To compare expression of a protein of interest from sample to sample, protein abundance is commonly normalized to a housekeeping gene. “When I see a large, dense band for the protein of interest or the housekeeping protein, I cringe,” says Gomes. That dense band usually means the protein of interest or housekeeping protein was no longer within the assay’s linear dynamic range. No accurate quantitative data can be extracted from such blots.

Another common reason for failure of quantitative western blots is flawed or incomplete protocols, according to Sean Taylor, the paper’s lead author and a Bio-Rad field application scientist (watch a video of him discussing the paper here). To address this, Taylor’s review pays special attention to experimental design and sample preparation and discusses proper definition of the linear dynamic range of protein loading, all key factors for generating meaningful quantitative western blot data.

Taylor also introduces more advanced concepts to improve reproducibility, simplify workflow, and reduce the time and cost of western blotting. One such technique is stain-free total protein normalization, which over the past year has proven superior to using housekeeping proteins or total protein staining to correct for loading errors.

With this article, Taylor hopes researchers now have a simple guide to ensure quantitative and reproducible western blot data for all research fields that rely on this technique.


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 2,500+ scientific posters on ePosters
  • More than 3,700+ 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 TechnologyNetworks.com 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.


Scientific News
Lemon Juice and Human Norovirus
Citric acid may prevent the highly contagious norovirus from infecting humans, scientists discovered from the German Cancer Research Center.
Signature of Microbiomes Linked to Schizophrenia
Studying microbiomes in throat may help identify causes and treatments of brain disorder.
Structural Discoveries Could Aid in Better Drug Design
Scientists have uncovered the structural details of how some proteins interact to turn two different signals into a single integrated output.
Protein Found to Play a Key Role in Blocking Pathogen Survival
Calprotectin fends off microbial invaders by limiting access to iron, an important nutrient.
Study Identifies the Off Switch for Biofilm Formation
New discovery could help prevent the formation of infectious bacterial films on hospital equipment.
How DNA ‘Proofreader’ Proteins Pick and Edit Their Reading Material
Researchers from North Carolina State University and the University of North Carolina at Chapel Hill have discovered how two important proofreader proteins know where to look for errors during DNA replication and how they work together to signal the body’s repair mechanism.
Protein Found to Control Inflammatory Response
A new Northwestern Medicine study shows that a protein called POP1 prevents severe inflammation and, potentially, diseases caused by excessive inflammatory responses.
X-ray Laser Experiment Could Help in Designing Drugs for Brain Disorders
Scientists found that when two protein structures in the brain join up, they act as an amplifier for a slight increase in calcium concentration, triggering a gunshot-like release of neurotransmitters from one neuron to another.
Team Identifies Structure of Tumor-Suppressing Protein
An international group of researchers led by Carnegie Mellon University physicists Mathias Lösche and Frank Heinrich have established the structure of an important tumor suppressing protein, PTEN.
Why We’re Smarter Than Chickens
Toronto researchers have discovered that a single molecular event in our cells could hold the key to how we evolved to become the smartest animal on the planet.
Scroll Up
Scroll Down
Skyscraper Banner

Skyscraper Banner
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
2,500+ scientific and medical posters
A library of 2,500+ scientific videos on LabTube
3,700+ scientific videos
Close
Premium CrownJOIN TECHNOLOGY NETWORKS PREMIUM FREE!