We aren’t alone in encouraging more rigorous Western blots. Many journal editors and funding agencies have voiced their support for producing better Western blots that are more quantitative and more accurate. Part of their reasoning is to enable better replicability for exciting new breakthroughs. Another part is how research needs to be considered credible by both peer researchers and the public to foster support and funding.
“Although the quantitative use of Western blotting is now widespread, published articles often lack the details of how Western blot results were quantified and how biological replicates were compared to obtain statistics.”
Scientific research is not for the sake of knowledge alone. We invest in scientific discovery, in part because we believe that one day something better will come of it. This is why the pharmaceutical and biotechnology industries rely so heavily on peer-reviewed published literature to find new ideas that might have wider application.
If you care enough to have your research built upon in the future, it is on you to make that research robust. If Western blots are a part of your research, being diligent about every experimental detail is imperative for other labs, clinics, and pharmacists who want to stand on your shoulders and take your discovery one step further. Improving research reliability ensures time and resources are spent validating robust data.
“Just because we can put numbers on an image does not imply that we should – a quantified biomolecule should relate directly to the true quantity of that biomolecule if it is to be meaningful.”
To get the very best Western blots, it all comes down to reducing variability. An imaging system and detection chemistry that matches these characteristics are ideal:
“Likewise, the production of light generated from fluorophores detected in the infrared spectrum not only improves quantification and accuracy, but facilitates normalization and comparative analysis months to years later without a loss of signal if membrane imaging is desired for future analyses. Therefore, infrared imaging has the potential to dramatically improve the efficiency of western blotting methods.”
Are your blots constant – or a constant source of worry? The best way to increase accuracy and replicability in your Western blots is to remove some variables from the equation. Each choice is a chance to reduce variability.
It may be easiest to start with how you compare blots and prepare them for publication. Choosing software designed specifically for analyzing Western blots makes your analysis reliable and effective.
Next, look at whether your signals are within the linear range of detection. This is affected by how you image your blots. Digital imaging is faster and much more consistent than exposing blots to film.
Then move to your chemistry. No matter what chemistry you use, make sure to validate antibodies before running an experimental Western blot. While ECL detection will work with some optimization, your signals will be more proportional and consistent with stable near-infrared fluorescence detection.
Finally, look at reducing sources of error in technique by choosing an appropriate loading control. Revert™ 700 Total Protein Stain is a good normalization strategy, because it has a wide linear range, high sensitivity, and the ability to correct for transfer errors without covalent modification of your target proteins.
LI-COR provides products, protocols, and support for Western blotting (and a range of other assays) that help reduce variability and increase replicability. Let us know how we can help you.Get in Touch