Bio-Rad Laboratories, Inc. has introduced the new Image Lab 4.1 software for use with the ChemiDoc™ MP imaging system. The new software automates multiplex analysis, providing a faster, more accurate alternative for quantifying protein expression levels via western blotting.
Proving that quantitative differences of a target protein are a result of biological differences and not experimental variability (such as unequal protein loading) is critical when conducting western blotting experiments. Typically, scientists comparing gene expression levels use a housekeeping protein (HKP) as a loading control with the assumption that HKP expression does not change based on treatment type or sample origin. However, both of these conditions can alter HKP expression, undermining its value as a normalization control.
Total protein normalization has been shown to be more stable than HKP normalization regardless of conditions or sample type (ref DOI 10.1002/pmic.200400941). Also total protein normalization saves time by eliminating the need for multiple probing steps or optimizing a multiplex blot, both of which are often required to normalize with housekeeping proteins.
Normalizing total proteins via stain-free detection, a capability provided only by Bio-Rad‘s ChemiDoc MP imaging system, offers advantages including speed, increased linearity, and wider dynamic range, over other total protein detection methods such as SYPRO Ruby and Ponceau S.
Image Lab 4.1 automates total protein normalization of multiplex fluorescent western blots by extracting image data directly from the ChemiDoc MP imager. Traditional image analysis methods require researchers to extract data into a spreadsheet program and manually manipulate data to produce a result. With Image Lab 4.1 software, however, researchers can deliver normalized intensities automatically by selecting detection and normalization channels, then clicking the Normalization button. Normalization is available with chemiluminescent blots as well as multiplex fluorescent blots.
Stain-free total protein normalization is part of Bio-Rad’s new V3 Western Workflow, which speeds the western blotting process and provides researchers with the reassurance that their immunoblotting process is working as expected. Using stain-free detection, scientists can visualize protein separation without staining, verify protein transfer to the membrane, and validate the results by total protein normalization.