Seeing is believing, but measuring is knowing

"We continue to screen all figures from accepted manuscripts,and we continue to find irregularities." The Journal of Clinical Investigations: Vol 119, Number 3, March 2009.

There is a lot of discussion in the scientific community about the credibility of western blotting data published in journals. It's no secret that questionable western blotting data is the most common reason for rejecting manuscripts. There is a recognized need by editors to improve the quality of western blotting protocols. To meet this challenge, the scientific community is investigating ways to address this.

Bio-Rad, as a member of the scientific community, has developed the V3 Western Workflow to increase confidence in western blotting data.

What is the V3 Western Workflow?

The V3 Western Workflow comprises a suite of products and Bio-Rad’s stain-free technology - a unique in-gel fluorescent compound that irreversibly binds to tryptophan residues enabling the direct visualization and quantification of proteins in gels and on blots. This novel workflow allows you to:

  • Visualize your gel and blot at each stage of the protocol
  • Get results faster with less hands-on labor
  • Accurately quantify your proteins of interest

How does the V3 Western Workflow ensure confidence in Western Blotting data?

Reliable Loading Controls

“There are several problems associated with this procedure [western blotting]: the expression of the control protein might in fact differ between samples, [and] the gel might be overloaded with regard to the control protein…” Journal of Proteome Research: 10, 1416-1419, 2011.

There are pitfalls when using housekeeping proteins (HKPs) as loading controls:

  • There is evidence that HKP expression levels change under different experimental conditions or sample types. Therefore, it is critical to validate that HKP levels remain consistent under specific experimental conditions when using them as loading controls for western blotting. This validation step adds a layer of complexity to the western blotting process.
  • Reliable western blotting data also requires the detection of both the target and loading control proteins within the linear dynamic range of the assay. Due to the high endogenous levels of traditionally used HKPs (GAPDH, actin, tubulin, etc.), these proteins are often overloaded, leading to oversaturation. Therefore, quantitative analysis based on this type of data is not reliable.

The V3 Western Workflow alleviates these problems by providing a more convenient and reliable total protein loading control using stain-free technology. Total protein loading controls are more reliable because

  • Total protein measurement is a true reflection of the amount of protein loaded for each sample
  • Total protein measurement exhibits a highly linear dynamic range in the amount of sample typically loaded for western blotting analysis (10–50 µg protein in a complex cell lysate) and accurately differentiates loading differences among samples

More Quantitative Data

"For quantitative comparisons, appropriate reagents, controls and imaging methods with linear signal ranges should be used." Nature Guide

When using film as a detection method, the linear dynamic range is narrow. When manipulating the exposure time, the expression level of a protein of interest may appear to vary. Therefore, it is necessary to validate the exposure time for film.

The V3 Western Workflow eliminates this issue by using stain-free technology with CCD imaging. Stain-free total protein loading control yields measurements within a linear dynamic range that housekeeping protein loading controls cannot match. Furthermore, the CCD camera imager has a broader linear dynamic range than film. The V3 Western Workflow combines these two advantages to provide you with more accurate quantitative data.

Raw Data Recorded At Each Step of the Workflow

"This may seem elementary, but keep the raw data. A well-annotated lab notebook can resolve problems very quickly....and further than just saving immunoblots - the same relates to histology and other data - print them out. Make multiple copies. Label the data carefully. Keep clear records." All data are not created equal, Ushma S. Neill, J Clin Invest. 2009; 119(3):424-424

The traditional western blotting workflow generates two pieces of data - the target protein blot and the loading control blot. These data may originate from different gels. With film imaging, the original data may be misplaced or mislabeled.

The V3 Western Workflow gives you at least four pieces of data from one gel. All data are in an electronic file format that is easily recorded and maintained. The four pieces of data are:

  • A stain-free gel image taken after the gel run
  • A stain-free image of the post-transfer gel
  • A stain-free image of the blot
  • A chemi blot image of the target proteins

Details about the image acquisition are included in image files and can be provided to journal editors upon request. With this transparency, the user achieves better control of the entire process, troubleshooting is simplified, and training new users is easier.

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