We have identified three parameters that have the greatest impact on quantitative western blotting and have detailed strategies to optimize them appropriately. Once proper normalization techniques are in place, the traditional western blot protocol will need some additional optimization before it can provide truly quantitative results. The R² of the plotted data for the entire range of total protein signal was determined to be 0.9990, whereas the R² for β-actin, GAPDH, and α-tubulin were 0.8851, 0.9438, and 0.8332, respectively. The blot shown was imaged using the Invitrogen iBright Imaging System, and Invitrogen iBright Analysis Software was used to quantitate the total protein signal in the lanes. 138000), and Invitrogen Goat Anti–Mouse IgG Alexa Fluor 680 secondary antibody (Cat.
The membranes were then washed 3 times for 2 min each with 20 mL of ultrapure water, followed by addition of Invitrogen primary antibodies against β-actin (Cat. The membranes were washed twice for 2 min each with 20 mL of ultrapure water and then labeled with 10 mL of a working solution of No-Stain Protein Labeling Reagent for 10 min. Proteins in the gels were transferred to mini PVDF membranes using the Invitrogen iBlot 2 Gel Transfer Device with iBlot 2 Transfer Stacks (P0 protocol for 7 min). Method: Invitrogen Bolt 4–12% Bis-Tris Plus gels were loaded with 10–50 μg of HeLa total protein lysate per lane and electrophoresed using MES running buffer. 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. Normalization is required to accurately assess differences in target abundance.