Are you doing all you can to ensure accurate fluorescent western blot quantitation?
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 this checklist of steps that can be taken to ensure your western blot quantitation is as accurate as it can be.
A quantitative western blotting checklist
Consistency is key!
- Have you treated your samples consistently, ensuring cells and tissues are thoroughly lysed, immediately placed on ice, and/or been treated with appropriate reagents such as protease inhibitors?
- Have you loaded similar amounts of total protein in each lane (within a factor of 2- to 5-fold)?
- Are you using a robust and consistent western blotting protocol (remember that you can reduce variability by using the same reagents for each blot or implementing lot-to-lot controls, and keeping your transfer conditions and incubation and wash times the same)?
- Are you normalizing to total protein?
- Have you validated the specificity of your antibodies?
- Have you verified that your system is linear?
The linearity of your signal can be affected by multiple factors. Here are steps you can take to ensure linearity and improve the accuracy of your quantitation:
- Are the highest and lowest amounts of your protein-of-interest in the linear range (note that you need to verify linearity with each protein-antibody pair)?
- Are you saturating your membrane?
- Have you optimized your primary and secondary antibody dilutions? Titrating your antibody against your protein-of-interest can reveal an antibody dilution that delivers the widest dynamic range.
- Have you optimized your image acquisition time to ensure you are in the linear range of your imager?
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:
Powerful and flexible western blot imagers
- Choose a cSeries Imager when you need an instrument that delivers it all—performance, bench-friendly footprint, and affordability. Available in a range of upgradable configurations that support imaging with NIR
fluorescence (lasers*), RGB fluorescence (LEDs), chemiluminescence, and UV and white light.
- Choose a Sapphire Biomolecular Imager when you need a scanning imager
and/or need the highest performance levels. Available in a range of upgradable configurations that support scanning/imaging with NIR fluorescence (lasers), RGB fluorescence (lasers), and phosphor imaging capabilities.
* Not sure why lasers make a difference? Fill out the form above and we’ll be
happy to answer your questions!
Reliable reagents that support accurate quantitation
- Normalize to total protein with AzureRed Total Protein Stain. Easily fitting into a typical western blot workflow, AzureRed provides a much wider dynamic range than commonly-used housekeeping proteins.
- Simplify western blotting and get greater consistency with a complete western blot kit like the AzureSpectra Fluorescent Western Blotting Kit. Available with either our Fluorescent Blocking Buffer or our Protein Free Blocking Buffer, these kits provide everything you need for two-color detection.
- Get low backgrounds and high specificity with AzureSpectra Fluorescent Secondary Antibodies. Ideal for sensitive detection, these secondaries are second to none.
- Quantitative chemiluminescence! And if you also do chemiluminescent blots, our Radiance Q chemiluminescent HRP substrates are optimized for quantitation, with a wide dynamic range and signal that’s stable for an hour or more.
Have questions? Need a quote? Want to schedule a demo? Complete the form above and someone will be in touch with you shortly.
To get a PDF version of this checklist, simply complete the form below. You can also add any questions about quantitative western blotting or request a quote using the comment field in the form.