Issue link: https://resources.nanotempertech.com/i/1526380
1 9 A P P L I C A T I O N N O T E The data shown in the three examples above highlights the importance of screening a wide range of copolymers for finding the optimal nanodisc to solubilize and stabilize membrane proteins. Such samples are suitable for downstream applications such as cryo-EM, enzyme assays, or protein binding studies. The specific characteristics of each copolymer class may confer advantages to a particular membrane protein, however there is no definitive rule that can be universally applied to identify the optimal candidate. Due to its high throughput and low sample consumption, the Prometheus Panta can be used in the miniaturized expression and purification protocol co-proposed by Cube Biotech and the Imperiali lab to quickly screen a variety of copolymers that will confer optimal properties for both thermal and colloidal stability of membrane proteins embedded in native nanodiscs, allowing for further successful cryo-EM structure determination (2) . In particular, isothermal DLS measurements utilizing as little as 10 µL of sample can be employed to initially efficiently triage potential copolymers in accordance with the sizing information, without the risk of false-positive results due to the heterogeneity of the sample or false-negative results due to proteolysis which occur in the SDS-PAGE. Furthermore, using the same 10 µL of sample, the combined measurements of nanoDSF, DLS, and backreflection across a thermal ramp from the Prometheus Panta give additional insight into the conformational stability of the protein and the overall colloidal stability of the nanodiscs, thus narrowing down the selection. We have demonstrated that the NativeMP TM Platform offered by Cube Biotech, combined with the time efficient, miniaturized expression and purification of the membrane proteins, and the use of the Prometheus Panta instrument for screening and characterization of the different native nanodiscs represents an efficient workflow to find optimal conditions for cryo-EM experiments with membrane proteins, minimizing the amount of time and sample needed. Summary