How Do I Validate My Western Blot Linear Range?

Validating your linear range is important in accurately detecting proteins in a Western blot. There are two primary steps in validating the linear ranges of your target protein and internal loading control (ILC): protein concentration assay and serial dilution. Empiria Studio^® Software can then utilize that information and find the combined linear range (CLR) for the target and ILC.

Protein concentration assay

To estimate the concentration of proteins in your sample, a bicinchoninic acid assay (BCA) or another colorimetric assay is key. It can help you determine the appropriate amount of sample to load. However, keep in mind that concentration assays cannot account for the variability that might occur during the steps of a Western blot protocol.

Serial dilution

Next, use a serial dilution to determine the linear range for the target and ILC. (Need a quick refresher on how to do a dilution series? Check out the “Dilutions and Calculations” learning path in the Lambda U education portal.) Their combined linear range is important to your experiment because it helps determine how much sample you should load to produce a linear signal response for the target and ILC.¹

Prepare and load a gel with two-fold serial dilutions of the sample.
Perform a Western blot detection and quantify the signal from your target and ILC.
Plot the target signals to determine where the signals accurately reflect protein abundance.

Empiria Studio software

Now that the linear ranges of your target and ILC have been validated, their combined linear range needs to be addressed. Fortunately, Empiria Studio Software makes this part simple. Its easy-to-follow workflow guides you through normalization, replicate comparison, validation methods, and data sharing. It can help validate the combined linear range and determine how much sample to load. You are then ready to continue with your experiment and get reliable Western blot results.

To easily validate the combined linear range and improve your research process, try Empiria Studio Software for a free 30-day trial.

**Figure 1. Viewing the combined linear range graph in Empiria Studio.** Overlapping areas of green in the color bars indicate the available range of sample loading for accurate detection of the target and ILC. The middle of this range should be an appropriate starting point for sample loading. In this example, that midpoint is approximately 5.5 µg of sample protein.

References:

Determining the Linear Range for Quantitative Western Blot Detection. Biocompare. Accessed 26 July 2019.