The Simple Western™ system allows for an automated, gel-free, and quantitative alternative to the traditional western blot. The technology employs capillary electrophoresis to separate the proteins in a sample by size, eliminating the hands-on processing during a traditional immunoblot and generating robust data in as little as 3 hours.
Utilization of our Simple Western™ Jess instrument improves the sensitivity, reproducibility and quantitative accuracy when compared to other legacy immunoblot methodologies.
Assays can be performed across three separate weight ranges, 2-40 kDa, 12-230 kDa, and 66-440 kDa with as little as 3 µL of sample and protein concentrations as low as 0.1 µg/µL. Up to 24 samples can be analysed per run, detecting multiple protein targets simultaneously through multiplexing of the chemiluminescent, NIR, and IR channels.
Additionally, to obtain further information from a single sample, RePlex™ reagents can be deployed to permit re-probing with new antibodies or total protein quantification for inter-run comparisons.
Figure 1: Schematic of capillary size-based separation immunoassay carried out by the Simple Western Jess. Data showing signal generated by two targets in the chemiluminescent (black) and NIR (red) channels in a single capillary with a representative pseudo image.
The Simple Western™ technology has been used in our hands to accurately quantify protein expression, determine DC50 and Dmax (degradation) constants in targeted protein degradation (TPD) molecule development, analyse biomarkers from tissue samples (ex vivo), and understand cell signalling pathways or therapeutic mechanisms of action. Once a robust assay set up has been achieved, the plate preparation can also be automated using our liquid handling robots.
Figure 2: Graphical workflow demonstrating key steps from sample to data generation using the Simple Western Jess instrument.
Cell lysates can be prepared from a multitude of sources, including cell lines, primary cells, and ex vivo tissue samples. The protein concentration is determined to ensure equal protein loading and direct comparison between samples before the Simple Western Jess instrument separates the proteins by size and probes for the target(s) of interest.
The example below demonstrates the robust and reproducible concentration dependent degradation of CDK9 with the SNS-THAL-032 PROTAC molecule, revealing a DC50 of 16. 1nM ± 7.6 nM and Dmax of 99%.
Figure 3: Concentration dependent degradation of CDK9 by SNS-THAL-032. The area under the curve for CDK9 is normalized to that of Actin before being adjusted to the Vehicle (DMSO) control.
For more information on the Simple Western™ technology and how it can be applicable to your therapeutic development pipeline check out our case studies or reach out to one of our expert scientists.