Steps you can take to get quantitative data from chemiluminescent western blots
While fluorescent detection used to be the most reliable way to obtain quantitative data from western blots, chemiluminescent detection can also deliver quantitative information. Here are a few things you can do to maximize the quality and reproducibility of chemiluminescent westerns so that you generate reliable, quantitative data.
The keys to quantitation
As with any quantitative study, consistency is the key to getting good results. Samples need to be handled the same way, and reagents, protocols, and workflows should be pre-validated and as identical as possible from run to run. But in addition to these general best practices, there are some steps you can take specific to chemiluminescent detection that will support generation of quantitative data.
1. Maximize your linear dynamic range by using a digital imager
Quantitation is only possible when the data you generate is linear, which is one of the drawbacks to using film for chemiluminescent detection. Fortunately, modern digital imagers far surpass the ~2-log dynamic range film offers, with Azure Biosystems’ cSeries™ Systems and Sapphire™ Biomolecular Imagers offering a dynamic range of up to ~6-logs.
Purified transferrin serially diluted and detected with a chemiluminescent western using Azure Radiance Substrate.
2. Maximize your linear dynamic range by using a long-lasting, quantitative ECL substrate
Not all ECL substrates are created equal. Azure’s Radiance ECL substrates—Radiance and Radiance Q—are designed with quantiation in mind. With a signal that persists for more than hours instead of minutes and a wide linear dynamic range, Radiance substrates are a great choice for quantitative chemiluminescent western blots.
Azure’s Radiance and Radiance Q chemiluminescent Western blot substrate is clearly better optimized for quantitation than the alternatives, with high sensitivity, a wider linear range than other chemiluminescent reagents, and a long-lasting signal.
3. Normalize chemiluminescent westerns to total protein
With journals and western blot experts recommending that blots be normalized to total protein rather than a housekeeping protein1-4, normalizing chemiluminescent western blots no longer requires stripping and re-probing the blot or running multiple blots if you use AzureRed Total Protein Stain. Compatible with both fluorescent and chemiluminescent detection, AzureRed Total Protein Stain is an efficient and robust way to normalize data for quantitative chemiluminescent westerns.
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1. Moritz CP. Tubulin or not tubulin: heading toward total protein staining as loading control in Western blots. Proteomics. 2017;17:1600189.
2. Thacker JS et al. Total protein or high-abundance protein: which oers the best loading control for Western blotting? Anal Biochem. 2016;496:76-78.
3. McDonough AA et al. Considerations when quantitating protein abundance by immunoblot. Am J Cell Physiol. 2015;308(6):C426-C433.
4. Fosang AJ, Colbran RJ. Transparency is the key to quality. J Biol Chem. 2015;209(50):29692-29694.