Azure Biosystems
A quick overview of the steps you can take to ensure your Western blots are quantitative. This free guide includes a troubleshooting section and tear-out quantitative Western blotting checklist.
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.
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. These features and more are available in the Azure Biosystems Imaging Systems and Sapphire™ Biomolecular Imager, including a dynamic range of up to ~6-logs.
Not all ECL substrates are created equal. Azure’s Radiance ECL substrates—Radiance and Radiance Q—are designed with quantitation 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.
With journals and western blot experts recommending that blots be normalized to total protein rather than a housekeeping protein 1-4, normalizing chemiluminescent Western blots no longer requires stripping and re-probing the blot or running multiple blots if you use AzureRed Total Protein Stain.
AzureRed Total Protein Stain is an efficient and robust way to normalize data for quantitative chemiluminescent Westerns. AzureRed is compatible with both fluorescent and chemiluminescent detection.
Getting quantitative data from your Western blot takes more than just a fluorescent secondary and digital imager. You need to consider your sample prep, your normalization method, and the linearity of your signal. To help keep your quantitative Western blot workflows on track, we’ve created a checklist of steps that can be taken to ensure your Western blot quantitation is as accurate as it can be.
Check out this publication from Boys Town National Research Hospital that imaged chemiluminescent Western blots with an Radiance HRP Substrate using an Azure imaging system.
With products that offer sensitive detection, wide dynamic range, easy-to-use features, and lot-to-lot consistency Azure Biosystems expertly streamlines and supports your quantitative fluorescent Western blot workflows.
