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Figure 1 shows the advantage of using No-Stain Protein Labeling Reagent as a substitute for traditional HKPs, which can easily become oversaturated. Once labeled, total protein can then be used to normalize the signal of your target protein. One TPN method utilizes Invitrogen No-Stain Protein Labeling Reagent, which covalently labels the total protein loaded in each lane with a fluorescent label. An accurate loading control should display a linear relationship between sample load and signal intensity in all experimental conditions.Īn alternative normalization method that is growing in popularity is total protein normalization (TPN), which normalizes the target signal with the total amount of protein loaded in each lane. With appropriate calculations, sample signals can be accurately normalized to housekeeping proteins designated as internal loading controls to obtain quantitative western blot data. It is important to avoid signal saturation in the normalization controls as well as the target protein, or accurate quantitation will not be possible. The relative intensity becomes nonlinear in shape and levels off, indicating virtually the same intensity for higher and higher amounts of protein. They include β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and α-tubulin, which have been shown to become saturated at common lysate loading amounts (e.g., 30–50 μg/well). Figure 1 shows some common normalization controls known as housekeeping proteins, or HKPs. When a chemiluminescent signal becomes saturated, the ability to relate signal intensity to protein abundance is lost.

When transitioning your western blotting experiments from qualitative to quantitative analysis, the most important attribute to address is signal saturation. Choosing the correct normalization method for your quantitative western blot is critical for obtaining reliable and reproducible results. Normalization corrects for unavoidable errors that occur during the western blot process, including sample loading or effects from electrophoresis, transfer, or sample concentration. Normalization is required to accurately assess differences in target abundance.