Negative stain electron microscopy (EM) is a longstanding method of sample validation. Take control of sample quality by direct visualization and discover unique protein properties unrivaled by other approaches. Negative stain analysis determines several aspects, such as aggregation and homogeneity, or examines potential oligomerization states, complex dissociation, and unfolding; even ligand binding of large complexes is visualizable with EM.
The method is rapid and requires no cryo-freezing or previous testing, making it an optimal answer to several questions. With low sample amounts (0.1 mg/ml, 30 µl) and rapid processing times, sample information has never been more accessible.
Aggregation and heterogeneity in samples can lead to unreliable functional results. Determine unwanted sample properties, save time ruling out problems, and decide on optimal buffer conditions. Screening samples in different buffers by EM quickly rules out flaws and pinpoints problem areas unseen in traditional methods, such as SDS-PAGE.
Proteins form oligomerization states between larger subunits and complexes. These states can be difficult to discern, and while many methods aid in the discovery of oligomerization, EM is the only method that provides visual proof simultaneously with quality control. Determine whether your complex forms the tetrameric state you suspected, or whether dimers-of-dimers were the reason for unexpected results.
Proteolysis targeting chimeras (PROTAC) are increasingly attractive as potential therapeutic agents. Negative stain TEM can determine the successful binding of E3 ubiquitin ligase and localize the general binding area with 2D views from multiple protein orientations. It is a great tool for insight into novel agents, and a stepping stone into 3D structure determination by cryo-EM. Negative stain analysis already covers the initial screening process, meaning the sample is immediately ready for cryo-screening and a high-resolution future.
Reconstitution into detergent or membrane-mimicking systems, such as nanodiscs, can take a lot of effort to optimize. Visualize protein particles with negative stain EM, and determine successful reconstitution while analyzing aggregation between different samples so you can save time by selecting the optimal conditions and ratios. We can measure particle diameter for sample comparison and to determine disc size.
We prepare everything else! We receive your sample – optimize and test it, before returning the data in a personalized way that suits you.
Get insight into detailed parameters with sample amounts of only 30-50 μl (0.01 mg/ml).
Get first results within 2 weeks.
Receive a full overview in an extensive report prepared by our cryo-EM experts, highlighting any details of interest.
