Bio-Rad Imagers Offer
Time Saved
Reduce your western workflow from 16 hours to 5 hours with Stain-Free technology.
Broad Dynamic Range
Capture 4 orders of magnitude in a single exposure.
Simplicity
Three clicks from gel load to data export.
skip to main content
Brilliant Western Blot Images. Zero Complexity.
Go from gel to publication-ready data in minutes, whether you’re looking for routine DNA documentation or advanced multiplex western blot analysis.
Speak to an Imaging Expert
Tired of Saturated Bands and Manual Bottlenecks?
Bio-Rad’s imaging solutions eliminate the guesswork of traditional film and the complexity of multi-step staining. Get publication-quality, quantitative results even from your most challenging low-abundance samples.
- Inconsistent data between blots?
- Faint bands lost in the background?
- Overnight incubations slowing down your productivity?
Systems Built for Every Lab
Frequently Asked Questions
When you request a demo, a Bio‑Rad imaging specialist will reach out to learn about your research and imaging needs. Based on your workflow, they will explain the most relevant capabilities of the Bio‑Rad imaging system based on your needs—such as gel imaging, chemiluminescent western blotting, fluorescent detection, and image analysis—as well as highlight additional capabilities, including Stain-Free gel technology, that can improve your overall workflow.
The demo is designed to help you understand:
- How the system supports your specific applications (e.g., gels, western blots, normalization workflows)
- What differentiates the imager in terms of ease of use, workflow efficiency, and data quality
- Which configuration is the best fit for your lab and budget
- Which aspects of the Stain-Free total workflow will yield time- and cost-savings for you
There’s no obligation to purchase; requesting a demo is simply a way to explore whether the system is right for your research before making a decision.
Request a Demo
What is Stain-Free Imaging Technology?
Stain-Free technology uses a polyacrylamide gel containing a proprietary trihalo compound that makes proteins fluoresce directly in the gel after a short ultraviolet (UV) photoactivation (Figure 1). This trihalo compound covalently binds to tryptophan residues, enhancing their fluorescence when exposed to UV light. As a result, you can immediately visualize proteins in gels or on blots. Stain-Free technology enables the detection of proteins at levels as low as 10-25 ng. A Bio-Rad Stain-Free-enabled imager is required.
Watch a video about the benefits of Stain-Free technology.
The Stain-Free workflow takes only 5 hours to complete, versus 16 hours for traditional western blotting. In addition to saving time, Stain-Free technology also allows you to assess transfer efficiency and collect other information quickly and easily during the western blotting process.
Is Stain-Free technology as sensitive as Coomassie blue stains?
Stain-Free gel data are comparable to data from gels stained with traditional dyes, with a few exceptions. In general, the sensitivity of Stain-Free technology is equal to that of Coomassie Brilliant Blue (CBB) for proteins containing low-to-medium levels of tryptophan. However, for proteins with higher tryptophan content, Stain-Free technology provides much higher sensitivity than CBB (Figure 2).
The limit of detection for the Stain-Free gels is 8-28 ng, similar to that of silver stains (0.6-1.2 ng), while Coomassie Brilliant Blue R-250 Staining Solution can detect protein amounts of at least 35-50 ng. Some fluorescent stains detect proteins below the 1 or 0.5 ng limit. Stain-Free gels have more reproducible data with smaller coefficients of variation compared to CBB or silver stains (McDonald et al. 2008, McDonald 2009).
The linear dynamic range for protein quantitation is the range where the signal intensity on a blot proportionally increases with the increase in protein load. Ideally, the protein load should fall within the quantitative linear dynamic range of the antibody used for its detection (Taylor and Posch 2014). Stain-Free gels provide a predictable linear dynamic range of 10-80 µg of total protein load from cell or tissue lysates at a higher range of protein load (Figure 3A) and from 1-20 µg at a lower range (Figure 3B) (Taylor et al. 2013, Hammond et al. 2013).
The complete transfer of proteins to the membrane is critical in western blotting. Traditional verification methods like Ponceau S, SYPRO Ruby, and other blot stains are more time-consuming than the Stain-Free method. Verifying protein transfer from gels to membranes using a Stain-Free-enabled imager can be accomplished in as little as 2 minutes. Unlike with traditional dye-based techniques, the observed intensity of the bands does not depend on the duration of staining or destaining (Figure 4).
Proteins that have passed through the Stain-Free workflow are compatible with many downstream applications such as western blotting, mass spectrometry, and chromatography (Figure 5). The Coomassie dye-based method does not allow the same gel to be used for transfer during western blotting or for mass spectrometry. This compatibility streamlines the workflow and enables total protein normalization and quantitation.
Analyze with Certainty on a PC or Mac
Automated lane and band detection, molecular weight determination, and normalization are just a few clicks away with Image Lab Software.
Image Lab Software Introduction
Learn the basics of Image Lab Software for ChemiDoc and Gel Doc Imaging Systems. Find out how to acquire, display, edit, and analyze gel and blot images and then display and export data. This step-by-step webinar is divided into chapters so that you can quickly find the information that you need.
Watch a video tour of the ChemiDoc MP in action.
Watch a video tour of the ChemiDoc Go in action.
Reliable assessment of changes in target protein expression levels requires measuring both the target protein and the loading control protein in their linear dynamic ranges for immunodetection. Traditionally, housekeeping proteins like actin, β-tubulin, or GAPDH have served as loading controls and in data normalization (Figure 10). Unfortunately, housekeeping proteins (HKPs) are usually highly expressed, whereas target proteins are often expressed in low abundance. In the HKP approach, large amounts of cell lysates may need to be loaded to detect target proteins, which can result in the overloading of HKPs and yield oversaturated reference bands out of their linear range.
Furthermore, HKP expression levels may not be constant but instead may vary with different experimental treatments and other factors. Stain-Free technology eliminates these issues by quantifying total proteins on the same blot as the proteins of interest. This method of data normalization, called total protein normalization (TPN), provides more accurate protein quantitation and western blotting results than traditional HKP approaches.
In this method, the total density for each lane is measured from a blot, and a lane profile is obtained. Specialized software from Stain-Free-enabled imagers can interpret the data from the lanes in three dimensions so that the same lane profile data can be viewed as a three-dimensional peak. The background is adjusted in such a way that the total background is subtracted from the sum of the density of all the bands in each lane.
Several major academic publishers have recently revised their editorial guidelines for data publication, emphasizing reproducibility and quantitation criteria. For example, the Journal of Biological Chemistry (JBC) provides guidelines for collecting and presenting data that specifically address quantitative western blot publication, guidance on normalization methods and supporting data, and concerns regarding HKP in protein normalization (see Table 1).
Further Reading
Nature Portfolio. Image integrity and standards. https://www.nature.com/nature-portfolio/editorial-policies/image-integrity, accessed April 26, 2024.
Fosang AJ and Colbran RJ (2015). Transparency is the key to quality. J Biol Chem 290, 29,692-29,694.
McNutt M (2014). Journals unite for reproducibility. Science 346, 679.
Yadav G and Oh K (2018). Defining the new normal in quantitative western blot data. https://www.bioradiations.com/defining-the-newnormal-in-quantitative-western-blot-data, accessed April 26, 2024.
Using Image Lab Software for Total Protein Normalization on Western Blots
Get an in-depth look at how to normalize your multiplex fluorescence western blots to the total protein signal. This video covers manual adjustment of lanes and bands, correction for blot background, and how to export data for publication or further analysis of your results.
Stain‑Free imaging is optional. Bio‑Rad imagers support a wide range of applications, including traditional gel imaging, chemiluminescent western blotting, colorimetric detection, and fluorescent workflows.
Stain‑Free technology is a unique option for researchers who want to streamline their western blot workflow and normalize to total protein, but you can continue using your existing gels, stains, and detection methods if you prefer.
Many labs start with their existing workflows and later choose to add Stain‑Free imaging as their needs evolve.