Total Protein Normalization using RePlex and NIR Detection

Housekeeping proteins are unreliable standards for protein normalization (PN). With Simple Western Technology, total protein normalization (TPN) is fast and reproducible, even with high concentration lysate samples. This protocol describes TPN using RePlex and NIR detection, allowing total protein detection following RePlex to remove antibodies from an immunoassay with chemiluminescent or fluorescent probes. TPN using RePlex and NIR detection is linear for high sample concentrations (0.5–2.5 mg/mL), facilitating the analysis of low abundance targets. Like all Simple Western assays, this method is fully automated, and results are ready in just a few hours.