Corporate Banner
Satellite Banner
Technology
Networks
Scientific Communities
 
Become a Member | Sign in
Home>News>This Article
  News
Return

Independent Study Reports V3 Western Workflow™ with Stain-Free Technology Yields Superior Western Blotting Results

Published: Tuesday, October 16, 2012
Last Updated: Tuesday, October 16, 2012
Bookmark and Share
Scientists at Flinders University in Bedford Park, Australia recently demonstrated the superiority of a method employing Bio-Rad’s stain-free technology to use total protein as a loading control in semiquantitative western blotting.

In their report in Analytical Biochemistry, published online on September 15, Dr. Alex Colella and his colleagues describe how the stain-free approach allowed them to assess the quality of both electrophoresis and western transfer before committing to subsequent steps of the western blotting procedure, providing more accurate blot data in fewer steps and less time in comparison with traditional loading control methods.

“Our research shows that Bio-Rad stain-free gels can improve the quality of data from semiquantitative western blotting experiments,” said Dr. Tim Chataway, head of the Flinders University School of Medicine Proteomics Laboratory.

To ensure accuracy in semiquantitative western blotting, one of the most important steps is the determination of the protein load in each gel. Loading controls are used to normalize for loading errors that can result from imprecise protein estimation, pipetting inaccuracy, or uneven protein transfer. Traditionally, researchers have relied on two loading control methods: reprobing of membranes with an antibody against a housekeeping protein such as β-actin, or the use of total protein stains such as SYPRO Ruby, Ponceau S, or Coomassie Blue. 

To use a housekeeping protein as a loading control, it must be established that the level of this protein remains constant when the experimental variable of interest is changed. In some cases, the expression level of a housekeeping protein may be too high or low in comparison with the protein(s) being studied, or such controls are unavailable or inappropriate, for example, when comparing the abundance of a specific protein in different tissue extracts in which the protein concentrations differ. While membrane protein stains do not suffer the same issues as housekeeping proteins, the method still adds significant cost and time to western blotting experiments.

Dr. Chataway’s results indicated that the stain-free approach provides a superior alternative to existing loading control methods because the quality of electrophoresis and western transfer results can be confirmed before applying antibodies for detection. Identifying any problems at each step allows researchers to target troubleshooting efforts. Scientists also have more time to optimize their workflows, as the stain-free method can cut the time required for this protocol in half, from two days to one.

Research Findings
Chataway and his team, led by postdoctoral researcher Colella, conducted a series of western blots using purified protein from rat retinas in amounts ranging from 10–40 µg. The proteins were separated by electrophoresis, transferred by blotting, and visualized using Bio-Rad’s V3 Western Workflow, a portfolio of stain-free enabled western blotting products that allows researchers to visually monitor proteins throughout all steps of an experiment. Researchers can simultaneously image their protein(s) of interest and total protein on each blot for easy normalization. Because the V3 Western Workflow requires fewer handling steps, the probability of procedural errors is lower than in the SYPRO Ruby and antibody methods.

 Dr. Chataway stated that stain-free gels and SYPRO Ruby staining yielded equivalent performance, but the use of stain-free gels reduced the time required to perform semiquantitative western blotting by 1.5 hours compared with SYPRO Ruby, and by one day compared with antibody detection of the housekeeping protein β-actin. The stain-free method also reduced overall costs by eliminating the use of expensive reagents.

“We found that stain-free technology provides a faster and more cost-effective alternative to traditional methodologies, producing an overall superior performance as a loading control compared with SYRPO Ruby or antibody,” said Dr. Chataway.


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.

Related Content

Presentations at the AACR Annual Meeting to Highlight Advances in Cancer Research Made Possible by Bio-Rad’s Droplet Digital™ PCR Technology
Tailoring Treatments and Tracking Mutations with Liquid Biopsies possible with ddPCR™ technology
Wednesday, April 22, 2015
Bio-Rad Reports Fourth-Quarter and Full-Year 2014 Financial Results
Company has introduced its S3e™ Cell Sorter during the quarter.
Saturday, March 07, 2015
Accurate Determination of Copy Number States for Multiallelic Copy Number Variations
Researchers use next-generation sequencing (NGS) and Bio-Rad’s Droplet Digital PCR (ddPCR™) technology to solve the technical challenge.
Tuesday, March 03, 2015
Awards for Bio-Rad's Protein Expression Products and Genomic Workflow Solutions
BioInformatics LLC has recognized Bio-Rad with two prestigious Life Science Industry Awards for “Best Protein Expression & Analysis Products” and “Best Workflow Solutions — Genomics.”
Monday, January 19, 2015
Quantitative Western Blotting Using Stain-Free Technology
Bio-Rad hosted two seminars at the EB 2014 in which KOLs discussed approaches to improve the accuracy of western blotting quantitation using Bio-Rad’s stain-free technology.
Friday, September 05, 2014
Bio-Rad Acquires Sequencing Technology Company GnuBIO
GnuBIO is a privately-held life sciences company that has developed a droplet-based DNA sequencing technology.
Friday, April 11, 2014
Royal Canadian Mounted Police to Use Bio-Rad’s KnowItAll® Spectroscopy Software for Analysis of Automobile Crime Scene Samples
Using the KnowItAll software to search multiple automobile paint layers found at a crime scene against the PDQ database, forensic scientists can determine the make, model, year, and manufacturing origin of the samples.
Sunday, March 30, 2014
Bio-Rad Partners with the U.S. Pharmacopeial Convention
Company announces the availability of a beta version of the USP Spectral Library™ for raw material identification in the food and drug industries.
Thursday, March 06, 2014
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
A Brief RT-qPCR “Field Guide” for MIQE Adherence
Bio-Rad’s Sean Taylor and Eli Mrkusich have published a practical guide for MIQE compliance.
Monday, December 09, 2013
Accurate Detection of Extremely Rare Mitochondrial DNA Deletions Associated with Aging
The study published in Aging Cell identifies a new tool to accurately analyze extremely rare mitochondrial DNA deletions associated with a range of diseases and disorders as well as aging.
Thursday, September 05, 2013
Researchers Develop Rapid, Cost-Effective Early Detection Method for Organ Transplant Injury
Chronix Biomedical and transplant expert Prof. Oellerich use Droplet Digital™ PCR to quantify early rejection biomarker.
Thursday, August 29, 2013
Accurate Quantification of NGS Libraries
A study has found that Droplet Digital PCR (ddPCR™) can be used as an accurate and precise method for quality control of NGS libraries.
Tuesday, August 20, 2013
New Biomarker Could Reveal Alzheimer's Disease Years Before Onset
A new study has reported the identification of what may be the earliest known biomarker associated with the risk of developing Alzheimer's disease (AD).
Wednesday, August 14, 2013
Producing Reliable Quantitative Western Blot Data
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.
Monday, June 17, 2013
Scientific News
The Changing Tides of the In Vitro Diagnostics Market
With the increasing focus in personalized medicine, diagnostics plays a crucial role in patient monitoring.
LaVision BioTec Reports on the Neuro Research on the Human Brain After Trauma
Company reports on the work of Dr Ali Ertürk from the Institute for Stroke and Dementia Research at LMU Munich.
NIH Study Shows No Benefit of Omega-3 Supplements for Cognitive Decline
Research was published in the Journal of the American Medical Association.
Less May Be More in Slowing Cholera Epidemics
Mathematical model shows more cases may be prevented and more lives saved when using one dose of cholera vaccine instead of recommended two doses.
Investigating the Vape
Expert independent review concludes that e-cigarettes have potential to help smokers quit.
NIH Launches Human RSV Study
Study aims to understand infection in healthy adults to aid development of RSV medicines, vaccines.
Researchers Discover Synthesis of a New Nanomaterial
Interdisciplinary team creates biocomposite for first time using physiological conditions.
Poor Survival Rates in Leukemia Linked to Persistent Genetic Mutations
For patients with an often-deadly form of leukemia, new research suggests that lingering cancer-related mutations – detected after initial treatment with chemotherapy – are associated with an increased risk of relapse and poor survival.
Flu Remedies Help Combat E. coli Bacteria
Physiologists from the University of Zurich have now discovered why the intestinal bacterium Escherichia coli (E. coli) multiplies heavily and has an inflammatory effect.
Marijuana Genome Unraveled
A study by Canadian researchers is providing a clearer picture of the evolutionary history and genetic organization of cannabis, a step that could have agricultural, medical and legal implications for this valuable crop.
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!