![]() No-Stain Protein Labeling Reagent outperforms common housekeeping proteins for signal linearity at higher protein loads. Total protein normalization using No-Stain Protein Labeling Reagent. No-Stain Protein Labeling Reagent provides a linear response curve with a wide dynamic range, enabling accurate normalization.įigure 1. 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. Use the table below to select the right loading control for your sample type.Normalization is required to accurately assess differences in target abundance. This ensures that you will be able to distinguish between the bands. Select a loading control that has a different molecular weight to the protein of interest. Loading controls can show if this effect has occurred and allows the variation in binding to be corrected for.Īs an example, to be published in many Nature journals, a paper must meet four general criteria, the first of which is that it must "provide strong evidence for its conclusions." This directly correlates to the necessity of controls to prove that results obtained are valid. This may result in more variation in binding compared to other areas of the gel. Proteins in the outer lanes of the gel are transferred to the membranes in a position close to the frame. This is an issue that is particularly important in signaling assays or experiments where a large number of lanes are being run at once. This is imperative when comparisons are made of the protein expression levels between samples. They can be used to check that there has been even transfer from the gel to the membrane across the whole gel. Loading controls have a second role as a control in western blots. When lanes have not been loaded evenly, loading controls can be used to quantify the protein amounts in each lane by using the density of the loading control band to correct for the differences in loading. Here the main reasons you need to be using loading controls: Loading controls can be used to normalize the levels of protein detected by confirming that protein loading is the same across the gel. Why choose our loading control antibodies?.Loading controls are absolutely essential to ensure the reliability of your data when comparing expression of a protein in different samples. Housekeeping genes are often chosen for use this purpose. Loading controls are usually proteins that exhibit high-level, constitutive expression in the cell type or sample you are studying. ![]()
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