Why choose total protein stain instead of loading control for Western Blot normalization?

Understanding the relative abundance of target proteins and effectively normalizing data are crucial aspects of Western blot analysis. While loading control antibodies have long been the gold standard, recent advancements have highlighted the potential advantages of using total protein stain (TPS) as an alternative approach.

Boster Bio provides Western blot services with different additional service options designed to enhance the reliability and credibility of your experimental results. One of the options we offer to customers is total protein stain for western blot normalization.

In this article, we explore the concept of total protein stain and its applications in Western blot analysis, discussing its benefits over loading control antibodies in different scenarios. We will also examine the limitations and considerations associated with TPS, providing insights into when and how it can enhance data interpretation and reliability.

What is total protein stain (TPS)?

Total protein stain (TPS) is an excellent tool in Western blot analysis. After transferring all the proteins onto the nitrocellulose membrane, total protein stain is applied as a fluorescent stain. Its purpose is to serve as a normalization tool, especially in quantitative WB experiments where knowing the absolute quantity of the target protein is not sufficient. In situations where you have twice the amount of the loading protein on the WB but lack something to compare it against, relying solely on the absolute reading becomes challenging. However, with the application of total protein stain, you can easily normalize the target protein signal to the total protein stain signal. This normalization allows you to obtain the relative level of the target protein of interest, providing valuable insights into its abundance.

What is an alternative for total protein stain?

A classic alternative to total protein stain is using loading control antibodies, which are antibodies that target universally present biomarkers known for their stable expression across different cells. These biomarkers, often referred to as housekeeping proteins, have been extensively studied and established as reliable loading controls in humans and mammals. Some examples of well-recognized loading control targets include GAPDH, β-actin, α-tubulin, histone 3, vinculin, and many others. By using these loading control antibodies, you can assess protein levels and compare across samples.

What are disadvantages of loading controls?

Issue 1: Easily saturated signal

The first issue with using loading control antibodies is that many of them are abundantly expressed in cells. This can become problematic when your target protein is not as abundant, and you need to load a higher amount of proteins to detect it clearly. In such situations, the signal from the loading control antibodies may become saturated, making it difficult to accurately normalize against them. When all loading control antibodies give the same saturated reading, it becomes challenging to effectively use them for normalization purposes.

Issue 2: Expression fluctuations due to cell treatments

The second issue arises when you apply any kind of treatment to your cells. While loading controls are generally assumed to be stably expressed, recent studies have highlighted that this may not always be the case. Some treatments can significantly impact the expression levels of loading control proteins, rendering the fluctuations significant. Consequently, many journals and peer reviewers are increasingly requesting researchers to provide evidence demonstrating that their specific treatment does not influence the expression of the chosen loading control. You will need to ensure the stability of the loading control's expression under experimental conditions to prove credibility and strengthen the reliability of your research findings.

How does total protein stain solve these issues?

The challenge resides in the fact that loading controls can easily reach saturation at high levels. Simply put, the linear range of loading control antibodies is relatively short. However, total protein stain offers a solution to this issue. Total protein stains are specifically designed to have long linear ranges, allowing for accurate quantification even when loading a significantly higher amount of protein. For instance, when using total protein stain, each lane of the Western blot can maintain a linear signal, even with a loading amount of 100-200ug per lane. This amount is approximately 20 times more than the typical loading quantity. To illustrate this advantage, we have provided data below comparing the loading control signal with the total protein stain signal (see Figure 1). You will observe that the loading control signal rapidly increases and then plateaus, while the total protein stain signal exhibits a gradual and linear rise. This feature is the greatest advantage of total protein stain in Western blot analysis.

TPS vs loading control signal graph

Figure 1. Total protein stain signal (blue) vs. loading control signal (orange)

What scenarios should you use total protein stain for?

Although loading control antibodies have some potential issues, they have traditionally been the gold standard in western blot analysis, trusted for their reliability in many scenarios. However, recent advancements have shed light on the advantages of using total protein stain in several experimental contexts. Let's discuss scenarios where total protein stain provides a superior approach for normalization and data interpretation.

Scenario 1: When you need to load extra protein because your target protein has low expression levels

When dealing with a target protein that exhibits low expression levels, it is necessary to load a higher amount of protein in your Western blot for detection. However, this poses a challenge for loading control antibodies as they can quickly reach saturation. Typically, when you load around 10ug of protein, loading control antibodies tend to work fine. However, once you increase the loading to around 20ug, the reliability of loading control antibodies starts to diminish. Their ability to accurately normalize protein levels becomes compromised, highlighting the limitations of loading control antibodies in situations where higher protein amounts are required.

Scenario 2: When you want to quantify and multiplex on the same gel

Another scenario where loading controls may prove insufficient is when you want to perform multiplex Western blotting using a Western blot imager with only two channels. In such cases, you aim to simultaneously detect two different targets on the same blot. However, with both channels already dedicated to different antibodies using distinct fluorescence signals, there is no additional channel available for a loading control antibody. This is where total protein stain can be utilized as a suitable alternative for the third antibody, which cannot be accommodated due to the channel limitations. By employing total protein stain, you can still obtain valuable normalization data despite the absence of a dedicated loading control antibody channel.

Scenario 3: When you aim to publish in prestigious journals or address clinical release assays

When you're looking to publish your paper in a prestigious journal, it's important to keep up with their requirements. Lately, some journals have been asking for total protein stain analysis or questioning how your treatment affects the expression levels of the loading control protein. In cases like submitting to established journals or dealing with clinical release assays, adding a total protein stain on top of the loading control antibodies can make your data more solid and reliable. Yes, it requires a bit of extra effort, but considering the overall significance of your research, the cost of performing a total protein stain is minimal. So, in these demanding situations, going the extra mile with a total protein stain is a great way to make your data more robust.

Limitations of the total protein stain

Total protein stain does require an additional step in the experimental process and incurs some more cost than loading control antibodies. However, apart from these considerations, there are minimal drawbacks associated with using total protein stain. Its benefits in providing a comprehensive analysis and normalization tool often outweigh the additional effort and cost.

Conclusion

Total protein stain has emerged as a valuable tool in Western blot analysis, offering distinct advantages in various experimental scenarios. While loading control antibodies have long served as reliable normalization tools, TPS addresses some of the limitations associated with their use. Total protein stain provides a solution for situations where higher protein amounts or multiplexing on the same gel are required, allowing for accurate quantification and normalization. Furthermore, as the scientific community and journals continue to diligently uphold the importance of rigor and reproducibility, incorporating TPS alongside loading control antibodies can strengthen data robustness and credibility. By weighing your specific experimental requirements and limitations, you can make informed decisions regarding the most appropriate normalization approach for your Western blot analyses.

At Boster Bio, we go the extra mile to ensure the reliability, accuracy, and credibility of your experimental results. That's why we offer a range of Western Blot services, including total protein stain for western blot normalization. With our total protein stain service, you can elevate the quality of your data by accurately normalizing your target protein signal to the total protein stain signal. Submit an inquiry today and schedule a meeting with our experts to discuss your project!