Metallothionein as a clonable tag for protein localization by electron microscopy of cells.

A benign, clonable tag for the localization of proteins by electron microscopy of cells would be valuable, especially if it provided labelling with high signal-to-noise ratio and good spatial resolution. Here we explore the use of metallothionein as such a localization marker. We have achieved good success with desmin labelled in vitro and with a component of the yeast spindle pole body labelled in cells. Heavy metals added after fixation and embedding or during the process of freeze-substitution fixation provide readily visible signals with no concern that the heavy atoms are affecting the behaviour of the protein in its physiological environment. However, our methods did not work with protein components of the nuclear pore complex, suggesting that this approach is not yet universally applicable. We provide a full description of our optimal labelling conditions and other conditions tried, hoping that our work will allow others to label their own proteins of interest and/or improve on the methods we have defined.

Front-of-Package Protein Labels on Cereal Create Health Halos.

Front-of-package protein labels are frequently added to breakfast cereals, aimed at increasing purchases by consumers who believe they would benefit from eating more protein. However, the overall nutritional compositions of such products are often not significantly better than similar products without protein labels, and may contain more sugar, sodium, and calories to improve taste. We conducted an online survey with 1022 US adults to examine consumer perceptions of two cereals (Special K Original and Special K Protein). Participants perceived Special K Protein as healthier and more nutritious, though less tasty, than Special K Original. Special K Protein was perceived as providing greater health benefits, such as being more likely to help them build muscle, stay healthy, and live longer. Many participants perceived no differences in the amounts of certain nutrients between the cereals, such as sugar (54.5%), sodium (59.2%), and calories (49.1%). Yet, when serving sizes are equalized to one cup, Special K Protein has more sugar, sodium, and calories than Special K Original. Though most participants reported viewing the Facts Up Front label, only 21.3% correctly chose Special K Original as having the larger serving size. This pattern of results suggests the presence of a health halo surrounding the protein-labeled product.

Single-molecule mechanics of protein-labelled DNA handles.

DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA-protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG)-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp) were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG) beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD) imaging control experiments revealed that quantum dot-streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein-DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular recognition in time-critical molecular motor studies.