BioTechniquesVol. 46, No. 2 CitationsOpen AccessCitationsNijsje DormanNijsje DormanSearch for more papers by this authorPublished Online:25 Apr 2018https://doi.org/10.2144/000113058AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail To the PointAnalysis of proteins is simplified by immobilization, which minimizes sample requirements and may also enable repeated measures. Tethering proteins to surfaces (e.g., chips) can, however, block reactions with substrates or subsequent labeling steps. One antidote is to place a tether between the protein and the surface to allow greater range of motion and improved accessibility. In a recent paper appearing in Langmuir, Jouzi et al. take a different tack and dramatically shrink the surface. Their “nanoneedle” was formed by dipping two microelectrodes into a carbon nanotube solution, applying a voltage, and slowly pulling the probes apart to form a 13-nm diameter needlelike structure. After covalently attaching streptavidin to the tips of the nanotubes, the needle was dipped into a solution containing biotin-conjugated alkaline phosphatase. To concentrate the proteins, the authors applied a positive voltage, and the conductive properties of the nanotubes attracted the negatively charged proteins to the nanoneedle tip, where the electrical field is strongest. Kinetic measurements showed a km comparable to solution-phase enzyme, indicating that steric hindrance is negligible. Although detection strategies are still insufficiently sensitive for single-molecule analysis, nanoneedles offer a means to concentrate precious protein samples for real-time analysis under near-native conditions.Jouzi et al. 2008. Nanoneedle method for high-sensitivity low-background monitoring of protein activity. Langmuir 24:10786-10790.Freeze FrameImaging of zebrafish embryos is a powerful technique for elucidating developmental pathways. However, each imaging modality usually requires a unique preparation process, making it difficult to visualize fluorescently labeled proteins in a sample that is also suitable for electron microscopy (EM). Looking for more flexibility, Nixon et al. examined a number of fixation conditions to find a process compatible with correlative light-EM, reporting their findings in Traffic. The method uses high-pressure freezing, with 0.7% low melting agarose in E3 as a cryoprotectant, followed by freeze substitution. Although this latter process employs uranyl acetate in acetone, it completely avoids osmium; subsequent low-temperature embedding is done with Lowicryl resin and UV polymerization. Ultrastructural features as assessed by EM were similarly preserved compared with an osmium-fixed, epon-embedded sample. Significantly, the new method faithfully captures notochord structures, which are typically not retained during traditional chemical fixation or a previously described resin-free frozen sectioning strategy. Equally high-quality preservation was apparent in electron tomographic reconstructions. The most significant strength of the new method, however, is the compatibility with green fluorescent protein (GFP) visualization and immunofluorescence, allowing correlation of fluorescence with immunogold labeling on EM sections. This method should be applicable to any fast-frozen material in which serial light microscopy and EM is desirable.Nixon et al. A single method for cryofixation and correlative light, electron microscopy and tomography of zebrafish embryos. Traffic [Epub ahead of print, Nov 20, 2008, doi:10.1111/j.1600-0854.2008.00859.x].Best and BrightestHaving too many options can make us unhappy, if we're not sure which choice is optimal. When shopping for readout methods for detection microarrays, the wrong selection can spur more than disappointment; it can also mean a false negative. Fortunately, an unbiased, comprehensive comparison of labeling and signal amplification methods appears in Molecular and Cellular Probes. As a test system, Vora et al. used a long oligonucleotide array for detection of Vibrio cholerae, probing test samples comprising biotinylated multiplex PCRs from dilutions of the organism's genomic DNA. Nine methods capable of detecting biotinylated DNA were compared with direct and indirect labeling. The major criterion was sensitivity, but intensity, signal-to-noise ratio, and cost/convenience were also assessed. At higher concentrations of DNA, all methods performed satisfactorily. Under more challenging conditions, sensitivity varied 100-fold, and there were substantial differences in signal intensity and background. If money and time are no object, resonance light scattering with anti-biotin antibody-conjugated particles gives the best results. However, at a cost-per-assay 18 times greater than the most economical option, it is unlikely to be the first choice. Indirect labeling and a modified tyramide signal amplification method were also sensitive, but reproducibility was an issue, most likely as a result of the many steps involved. Interestingly, the cheapest method—which uses NeutrAvidin and Cy5-labeled cowpea mosaic virus particles—scored well in many of the parameters examined. Other applications will have different demands, so the authors provide detailed evaluations of each strategy, helping readers decide what option is best for their particular system.Image reprinted with permission. © 2008 ElsevierVora et al. 2008. Comparison of detection and signal amplification methods for DNA microarrays. Mol Cell Probes 22:294-300.