(1093-B) Simple Western with Jess is even more simple with CyBio FeliX Liquid Handling

Abstract: Automated western blotting using ProteinSimple Jess can be versatilely used, e.g., for drug discovery. The Analytik Jena CyBio FeliX was used for protein quantification setup and Jess plate preparation, hereby significantly improving workflow efficiency. PROTAC THAL-SNS-032 and its target CDK-9 were used to demonstrate the capabilities to automate and simplify the Jess preparation procedure using the CyBio FeliX liquid handler. First, the anti-CDK9 antibody was optimized to ensure saturation and signal linearity, making data quantifiable. CyBio FeliX performs a serial dilution of assay cell lysates and prepares the Jess plate with varying antibody dilutions. Then, a BCA assay was prepared by the liquid handler to determine protein concentration in lysates obtained from PROTAC-treated cells and to provide a data file for the main PROTAC assay. Last, antibody optimization results and BCA assay data were combined to set up the Jess plates, again using the CyBio FeliX liquid handler for process automation. Jess data analysis confirmed linear protein concentration-dependent detection of CDK9 as well as consistent signals indicating saturation across antibody dilutions. Dose-dependent CDK9 degradation by SNS-THAL-032 was consistent across multiple experiments. The quality of the automated process executed by the CyBio FeliX is the same as that of the manual process, while saving a scientist up to 60 minutes of bench time per Jess run. In conclusion, it was demonstrated that the CyBio FeliX can be used to optimize antibody & sample protein concentrations and prepare ProteinSimple Jess plate for screening purposes.