Posts Tagged ‘western blotting’

Designing the Perfect Quantitative Western Blot

 :: Posted by American Biotechnologist on 04-02-2014

A methods article published yesterday 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.

Although western blotting is a well-established laboratory technique, it has recently come under fire as a quantitative method because extreme care must be taken when generating and interpreting the resulting data.

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.

Sean Taylor discusses the BioMed Research International paper he coauthored.

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 on the left). 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.

To read the open access research article, visit http://bit.ly/1kCAOcr.

For additional resources please consult Bio-Rad’s guide to Troubleshooting Western Blots.

Video: How to Perform Perfect Quantitative Western Blotting

 :: Posted by American Biotechnologist on 04-01-2014

What You Don’t See Truly Matters

 :: Posted by American Biotechnologist on 11-13-2013

All gels are not created equal when it comes to protein transfer. Mini-PROTEAN® TGX™ precast gels, together with the Trans-Blot® Turbo™ transfer system, offer you a complete solution for achieving fast electrophoresis and transfer times. In addition to speed, these innovative gels deliver maximum transfer of proteins onto membranes so you don’t see proteins left behind on the gel, ensuring quality western blotting results you can count on every time.

For more information visit Bio-Rad Laboratories TGX™ precast gel page.

Video Tutorial: Quantitative Western Blotting

 :: Posted by American Biotechnologist on 06-17-2013

Are you getting the most from your western blot data? Does Imaging technique matter? Film or Imager? In this video, we describe a methodology to obtain reliable quantitative data from chemiluminescent western blots using standardization procedures coupled with the updated reagents and detection methods. For the best resolution, watch the video in full screen at HD resolution.

Expect More From Your Chemiluminescent Substrate

 :: Posted by American Biotechnologist on 10-31-2012

Obtaining high quality chemiluminescent blot images with little optimization is a challenge when dealing with a wide range of protein loads and expressions.

Bio-Rad’s new Clarity western ECL substrate provides high sensitivity (see Figure below), long signal duration, and low background even at long exposures. This combination of bright, long signal with low background makes it the perfect choice for routine use on film and digital imagers, even when re-imaging and multiple exposures are required.

The Clarity western ECL substrate is compatible with any HRP-conjugated secondary antibody and is offered in 200 and 500 ml size kits.

Read more…