A rapid ceramide synthase activity using NBD-sphinganine and solid phase extraction.

Ceramides are synthesized by six mammalian ceramide synthases (CerSs), each of which uses fatty acyl-CoAs of different chain lengths for N-acylation of the sphingoid long-chain base. We now describe a rapid and reliable CerS assay that uses a fluorescent N-[6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl) (NBD) sphinganine substrate followed by separation of the NBD-lipid substrate and products using solid phase extraction (SPE) C18 chromatography. SPE chromatography is a quick and reliable alternative to TLC, and moreover, there is no degradation of either NBD-sphinganine or NBD-ceramide. We have optimized the assay for use with minimal amounts of protein in a minimal volume. This assay will prove useful for the analysis of CerS activity, which is of particular importance in light of the growing involvement of CerS in cell regulation and in the pathology of human diseases.

Drop-by-drop chemical reaction and sample introduction for capillary electrophoresis.

In this paper, we report a novel sample introduction and chemical reaction strategy by drop-by-drop inkjet injection for an electrophoretically mediated microanalysis (EMMA). This method makes it possible to achieve an on-line introduction of reactant solutions by alternately ejecting small plugs, with an overlapping region of the plugs for mixing the reactants by electrophoresis, supporting chemical reactions, followed by electrophoretic separation of the final compounds. As a proof-of-concept of the method, the EMMA of an inkjetted mixture of 4-fluoro-7-nitrobenzofurazan (NBD-F) and amino acids was carried out as a model chemical reaction. The product NBD-amino acids were quantified by detection with laser induced fluorescence. The optimal conditions for the procedure were: inkjet driving voltage: +40-44 V; pulse width: 20-24 µs; drop-by-drop injection of reactant solutions: alternately 2 drops × 25 times for the amino acid solution and the NBD-F solution; zone overlapping voltage and time: 3 kV and 2 s; incubation time after overlapping: 5 min; separation voltage: 18 kV. Under the optimized conditions, a significant enhancement in sensitivity and a sensitive quantitative analysis were realized. The results obtained were comparable with those using the off-line labeling method. This method is rapid, cost-effective, and readily automated for EMMA.

Brownian ratchets: molecular separations in lipid bilayers supported on patterned arrays.

Brownian ratchets use a time-varying asymmetric potential that can be applied to separate diffusing particles or molecules. A new type of Brownian ratchet, a geometrical Brownian ratchet, has been realized. Charged, fluorescently labeled phospholipids in a two-dimensional fluid bilayer were driven in one direction by an electric field through a two-dimensional periodic array of asymmetric barriers to lateral diffusion fabricated from titanium oxide on silica. Diffusion spreads the phospholipid molecules in the orthogonal direction, and the asymmetric barriers rectify the Brownian motion, causing a directional transport of molecules. The geometrical ratchet can be used as a continuous molecular sieve to separate mixtures of membrane-associated molecules that differ in electrophoretic mobility and diffusion coefficient.