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How Stain-Free Western Blotting Contributes to Laboratory Efficiency

Gloved hand placing a gel onto a western blot membrane in a blotting cassette.
Credit: iStock.
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Western blotting is a staple technique in many laboratories, yet the process is time-consuming and the numerous manual steps can introduce variability. Stain-FreeTM technology can help to streamline the western blotting workflow and offers advantages for increasing throughput as well as analyzing low-abundance proteins and post-translational modifications.

Technology Networks spoke with Harsha Rao, field marketing manager at Bio-Rad, to learn more about Stain-Free western blotting and the advantages of total protein normalization.

Karen Steward (KS): For readers who may be unfamiliar with the concept, can you explain how Stain-Free western blotting works?

Harsha Rao (HR): Stain-Free western blotting is a method that enables total protein normalization without the need for traditional staining and destaining.

With Stain-Free western blotting, protein separation is completed by electrophoresis and the gel is activated under UV light. This enables a fluorescent dye located within the polyacrylamide gel to bind an amino acid covalently within proteins in the gel, enabling in-gel visualization and visualization on the membrane post-transfer.

Depending on the specific protocol and the number of samples being analyzed, Stain-Free western blotting can save anywhere from a few hours to an entire day compared to traditional western blotting methods.

KS: What advantages do Stain-Free systems offer over traditional western blotting techniques? Are there any drawbacks?

HR: There are many advantages in using Stain-Free western blotting, and they extend beyond the actual assay procedure. The workflow is streamlined with the number of manual steps reduced. Additionally, using total protein normalization saves time in terms of data analysis and interpretation, contributing to overall efficiency in the laboratory.

These benefits are only available when researchers have access to a Stain-Free enabled imager and require activation of the gel within the imager prior to transfer to a membrane. While these are additional steps, they require minimal time (> 5 mins) and ensure a more reliable and visible guide of procedure success at multiple points throughout the process.

KS: How does the analysis and normalization of a Stain-Free western blot on Bio-Rad’s system compare to traditional analyses?

HR: Normalization methods differ between traditional and Stain-Free blotting with traditional methods often relying on housekeeping proteins (HKP) (e.g., actin or tubulin) as loading controls. HKPs are highly expressed, whereas target proteins may be present in low abundance, requiring a large amount of sample to be loaded to enable their detection. The challenge is this can likely result in the HKP being outside its linear dynamic range. This discrepancy is often not accounted for in western blot experiments.

By utilizing Stain-Free total protein measurement as the loading control, researchers can ensure that both target proteins and loading controls are assessed within their linear dynamic ranges in western blot experiments. This is because total protein normalization stains exhibit linearity within the common loading range of 10–50 μg of cell lysate per lane and this approach enables the measurement of both low- and high-abundance target proteins.

KS: Are there any application areas or sample types where the Stain-Free system is particularly helpful over traditional methods?

HR: Stain-Free technology outperforms traditional methods in various application areas and sample types, particularly where speed, sensitivity and reproducibility are crucial. Here are some specific scenarios where Stain-Free technology shines:

  • Time-sensitive experiments or high-throughput analysis: Stain-Free technology is exceptionally useful when rapid results are needed or in high-throughput experiments where processing large numbers of samples is essential. Its streamlined workflow makes it ideal for analyzing multiple samples in a time-efficient manner.
  • Low-abundance proteins: Stain-Free technology empowers total protein normalization, allowing for increased loading of lysate without the risk of exceeding linearity. This is particularly beneficial in cases requiring quantitation of rare or low-level proteins.
  • Post-translational modifications (i.e. phosphorylation): Researchers interrogating signaling cascades often use western blotting to measure phosphorylation states of target proteins. Using housekeeping proteins requires an additional channel for normalization in addition to the channels used for total target protein and for the phosphorylation state. By incorporating Stain-Free technology and total protein normalization, an additional channel can be allocated to measure a target protein or its phosphorylation state.

KS: Do you have any exciting developments in the pipeline that you’d like to share with us?

HR: We are excited to mention an upcoming launch of a new and compact ChemiDoc Go imager offering multiple detection techniques. We have improved our digital imaging capabilities without compromising sensitivity with the same high-quality images Bio-Rad is known for. Stay tuned to find out more in the coming weeks!

Harsha Rao was speaking to Dr. Karen Steward, Senior Scientific Specialist for Technology Networks.

About the interviewee

A head shot of Harsha Rao.

Harsha Rao is a dynamic professional in the life science industry with experience managing life sciences and diagnostics products. With a proven track record of spearheading successful product launches and engaging with customers across diverse sectors, she is driven by a passion to introduce and support life-saving products in the market. Currently serving as a field marketing manager at Bio-Rad, Harsha brings her expertise and dedication to driving advancements in healthcare.