Since its introduction in 1959, Ellman's reagent (5,5'-dithio-bis(2-nitrobenzoic acid)) has been the favorite reagent for spectrophotometric measurement of protein sulfhydryls. Meanwhile however, evidence has accumulated that many protein sulfhydryls give an incomplete reaction with Ellman's reagent, even during prolonged assay times. In the present study, the kinetic problem was solved by including cystamine as a "mediator" between the protein sulfhydryl and Ellman's reagent, as previously applied in an enzymatic thiol assay [9]. As an alternative, 4,4'-dithiodipyridine (DTDP) was used in place of Ellman's reagent. Due to its small size, amphiphilic nature, and lack of charge, DTDP quickly reacts with poorly accessible protein sulfhydryls, without any catalysis by cystamine. The DTDP method and the Ellman/cystamine method were both optimized for maximal sensitivity, minimal sample consumption (detection limit 0.2 nmol mL(-1), determination limit 0.6 nmol mL(-1)), and minimal assay time (5 min). In validation experiments, both methods gave identical results and the measured sulfhydryls/protein matched the expected values. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00216-002-1347-2.

This study provides a critical examination of protein labeling with Cy3, Cy5, and other Cy dyes. Two alternate situations were tested. (i) Antibodies were covalently labeled with Cy dye succinimidyl ester at various fluorophore/protein ratios and the fluorescence of the labeled antibodies was compared to that of free Cy dye. (ii) Fluorescent biotin derivatives were synthesized by derivatizing ethylenediamine with one biotin and one Cy3 (or Cy5) residue. The fluorescence properties of these biotin-Cy dye conjugates were examined at all ligand/(strept)avidin ratios (0

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The unique ability of living systems to translate biochemical reactions into mechanical work has inspired the design of synthetic DNA motors which generate nanoscale motion via controlled conformational change. However, while Nature has evolved intricate mechanisms to convert molecular shape change into specific micrometer-scale mechanical cellular responses, the integration of artificial DNA motors with mechanical devices presents a major challenge. Here we report the direct integration between an ensemble of DNA motors and an array of microfabricated silicon cantilevers. The forces exerted by the precise duplex to nonclassical i-motif conformational change were probed via differential measurements using an in-situ reference cantilever coated with a nonspecific sequence of DNA. Fueled by the addition of protons, the open to close stroke of the motor induced 32 +/- 3 mN/m compressive surface stress, which corresponds to a single motor force of approximately 11 pN/m, an order of magnitude larger than previous classical hybridization studies. Furthermore, the surface-tethered conformational change was found to be highly reversible, in contrast to classical DNA motors which typically suffer rapid system poisoning. The direction and amplitude of motor-induced cantilever motion was tuneable via control of buffer pH and ionic strength, indicating that electrostatic forces play an important role in stress generation. Hybrid devices which directly harness the multiple accessible conformational states of dynamic oligonucleotides and aptamers, translating biochemical energy into micromechanical work, present a radical new approach to the construction of "smart" nanoscale machinery and mechano-biosensors.