what is loading control in western blot

Western blot analysis is a widely used method for detecting and quantifying proteins. To ensure the reliability of the results, the use of loading controls is an important step for Western blot analysis. Loading control serves as a reference for the amount of protein loaded onto the gel, which helps to standardize the results and confirm the validity of the findings.

What is Loading Control?

Loading control is a protein that is used as a reference for normalizing the amount of protein loaded onto the gel during Western blotting. Loading control is typically a stable, constitutively expressed protein that is present in all samples and does not change under different conditions. Since the amount of protein in different samples can vary due to experimental variations or differences in protein expression levels, loading control helps to account for these factors and ensure data accuracy.

Types of Loading Controls

There are several types of loading controls that can be used in Western blotting. The most commonly used loading controls are housekeeping proteins, such as beta-actin, GAPDH, and beta-tubulin. These proteins are highly expressed in most cells and tissues and are considered reliable reference proteins for normalizing protein expression levels. Other loading controls such as histones, lamin, and tubulin can also be used depending on the experimental design and the specific protein of interest.

How to Use Loading Control?

The loading control should be run on the same gel as the protein of interest. The protein of interest is then normalized to the loading control using image analysis software. This will provide an accurate measurement of the relative protein expression level. It is important to select an appropriate loading control for the experimental conditions because different experimental treatments can affect the expression levels of different loading controls.

Conclusion

A loading control is an essential tool for Western blot analysis to ensure that the data obtained from the experiment is accurate and reliable. It is necessary to carefully choose a suitable loading control that is stable, constitutively expressed, and does not change under different experimental conditions. By normalizing the protein levels to the loading control, researchers can confidently compare the expression levels of different proteins among different samples and experimental conditions.