Objective

Many assays systems we work with on a daily basis are highly purified systems and balancing this with physiological relevance for screening is an ongoing challenge In this presentation we will share our experiences with the development of an assay and subsequent high throughput screen to identify modulators of sarcomere activity. The assay was based on the use of semi-crude cardiac muscle homogenate and compared with a reconstituted sarcomere preparation. From a technical perspective, we will cover some of the steps taken to overcome the challenges of liquid handling and detection conditions when working with viscous and particulate reagents in a 384-well assay format with a small assay window and the considerations for high throughput screening. Interestingly, the data obtained demonstrates how two very similar reagents used under the same assay conditions can provide different results in terms of sensitivity to activation and inhibition of the same biological pathway.