Concentrating On AAV Impurities With Ultrasensitive Total Protein Detection On Simple Western

TOTAL PROTEIN DETECTION WITH SIMPLE WESTERN

Impurities in protein products can be dangerous and impact efficacy. For example, protein impurities in final drug products could lead to undesirable immune responses in patients, so detecting total protein is critical for revealing impurities in preparative protein production. Traditional methods for total protein detection rely on SDS-PAGE with dyes like Coomassie Blue, SYPRO Ruby, or silver stain. However, SDS-PAGE requires large sample volumes, a lot of hands-on time, and it is poorly reproducible. Also, the use of staining dyes often comes with a lot of waste and can require specialized imaging equipment to which not every researcher has access.

Conversely, Simple Western™ assays on instruments like Jess and Wes from ProteinSimple offer fully automated protein separation and quantification with small sample volumes, and sensitive chemiluminescent-based immunodetection and total protein detection. While immunoassays on Simple Western allow target-specific detection, the Total Protein Detection Module allows for all proteins to be labeled and detected, which is ideal for monitoring impurities. Here, we show that total protein detection with Simple Western can be even more sensitive by using 5 times more concentrated biotin labeling reagent, resulting in protein detection that surpasses the sensitivity of protein stains like SYPRO Ruby. While SYPRO  Ruby requires at least 1 ng of protein for reliable detection,^1-2 Simple Western can reliably detect as little as 150 pg. This SYPRO Ruby-beating sensitivity improvement makes Simple Western well suited for the analysis of precious samples such as Adeno-Associated Virus (AAV) samples used in Cell & Gene Therapy workflows.