Effect of carbon cup aging on plasma zinc determination by flameless atomic adosorption spectrometry.
(33/796)
Determination of zinc in blood plasma by flameless atomic absorption spectrometry is discussed, with particular reference to the protocol required for the successful use of the Varian-Techtron Carbon Rod Atomizer. Cup aging is shown to be an important factor in limiting the precision of this analytical technique and ways of minimizing the problem are described. Matrix problems have also been encountered, which precluded the use of aqueous standard curves and the method of standard additions. We propose the use of plasma in preparing standard curves, the values for which are corrected for inherent plasma zinc, as a possible solution to the problem. (+info)
Microfabrication of individual 200 microm diameter transdermal microconduits using high voltage pulsing in salicylic acid and benzoic acid.
(34/796)
We describe an extension of semiconductor fabrication methods that creates individual approximately 200 microm diameter aqueous pathways through human stratum corneum at predetermined sites. Our hypothesis is that spatially localized electroporation of the multilamellar lipid bilayer membranes provides rapid delivery of salicylic acid to the keratin within corneocytes, leading to localized keratin disruption and then to a microconduit. A microconduit penetrating the isolated stratum corneum supports a volumetric flow of order 0.01 ml per s with a pressure difference of only 0.01 atm (about 10(2) Pa). This study provides a method for rapidly microengineering a pathway in the skin to interface future devices for transdermal drug delivery and sampling of biologically relevant fluids. (+info)
A method for parallel, automated, thermal cycling of submicroliter samples.
(35/796)
A large fraction of the cost of DNA sequencing and other DNA-analysis processes results from the reagent costs incurred during cycle sequencing or PCR. In particular, the high cost of the enzymes and dyes used in these processes often results in thermal cycling costs exceeding $0.50 per sample. In the case of high-throughput DNA sequencing, this is a significant and unnecessary expense. Improved detection efficiency of new sequencing instrumentation allows the reaction volumes for cycle sequencing to be scaled down to one-tenth of presently used volumes, resulting in at least a 10-fold decrease in the cost of this process. However, commercially available thermal cyclers and automated reaction setup devices have inherent design limitations which make handling volumes of <1 microL extremely difficult. In this paper, we describe a method for thermal cycling aimed at reliable, automated cycling of submicroliter reaction volumes. (+info)
High sensitivity automated sequence determination of polypeptides.
(36/796)
We report the development of a high sensitivity Edman method for use in the automated protein sequenator. With the use of a radioactive coupling reagent, [35S]phenylisothiocyanate, and minor modifications in the sequenator program, sequence analyses have been performed on nanomole quantities of protein. The radioactive phenylthiohydantoin derivatives produced in the degradation are identified at the 10 to 100 pmol level by two-dimensional thin layer chromatographic procedures with autoradiography and are quantitated by scintillation counting. This high sensitivity approach, which is about 100 times more sensitive than conventional automated Edman procedures, has allowed continuous amino acid assignments for 15 or more cycles on quantities of protein less than 5 nmol. It has been employed in the NH2-terminal sequence analysis of several proteins whose sequences were previously undetermined. (+info)
Single-cell dissection and microdroplet chemistry.
(37/796)
The unique roles of individual cells may be critical to the physiology of an organism. In such cases, micromethods are essential to elucidating the molecular biology, biochemistry and biophysics of the specialized cells or even subcellular compartments of the important cells. The great proliferation of micromethods testifies to their value and no single review can be comprehensive. This review therefore provides only a generalized overview of one approach, namely dissection that provides a pure sample for subsequent extraction and analysis by microdroplet chemistry. As a means of illustrating the utility of this approach, an application-study of the interaction of cytosolic malate concentration and guard-cell phosphoenolpyruvate carboxylase-is provided. (+info)
Use of octylbenzenesulfonate in place of dodecyl sulfate in SDS-polyacrylamid gel electrophoresis.
(38/796)
Sodium n-octylbenzene-p-sulfonate was successfully used in place of sodium dodecyl sulfate in SDS-polyacrylamide gel electrophoresis. This modification of the usual technique made it possible to scan the polyacrylamide gel for the distribution of the surfactant by measurement of UV absorption. It was found that a band electrophoresed in advance of the complexes formed between protein polypeptides and the surfactant. The band could be observed even in the absence of protein polypeptide, and was ascribed to micelles derived from surfactant added in excess to the sample solution. The complexes were found to be detectable by scanning at 261 nm, usually with better sensitivity than by scanning at 280 nm. Thus, this modification of SDS-polyacrylamide gel electrophoresis is useful both to investigate what is going on in the gel during electrophoresis and to improve the sensitivity of detection by UV scanning of protein complexes. (+info)
Rapid screening of the aglycone specificity of glycosidases: applications to enzymatic synthesis of oligosaccharides.
(39/796)
BACKGROUND: Retaining glycosidases can catalyse glycosidic bond formation through transglycosylation from a donor sugar to an acceptor bound in the aglycone site. The aglycone specificity of a glycosidase is not easily determined, thereby complicating the choice of the most appropriate glycosidase for use as a catalyst for transglycosylation. We have developed a strategy to rapidly screen the aglycone specificity of a glycosidase and thereby determine which enzymes are best suited to catalyse specific transglycosylation reactions. RESULTS: The reactivation, or turnover, of a glycosidase trapped as a fluoroglycosyl-enzyme species is accelerated in the presence of a compound that productively binds to the aglycone site. This methodology was used to rapidly screen six glycosidases with 44 potential acceptor sugars. Validation of the screening strategy was demonstrated by the identification of products formed from a transglycosylation reaction with positively screened acceptors for four of the enzymes studied. CONCLUSIONS: The aglycone specificity of a glycosidase can be rapidly evaluated and requires only an appropriate fluorosugar inactivator, a substrate for assay of activity and a library of compounds for screening. (+info)
Liquid-chromatographic analysis for neutral carbohydrates in serum glycoproteins.
(40/796)
We describe a sensitive, reproducible procedure of analysis for the six neutral carbohydrates in glycoproteins, by high-resolution anion-exchange chromatography. As many as 16 neutral carbohydrates can be separated by elution with a concentration gradient of boric acid (pH 7, 67 to 672 mmol/liter). The carbohydrates are detected with a cerate oxidimetric detector system, which monitors the fluorescence of Ce3+ produced by the reaction of the eluted constituents with Ce4+. Sensitivity to 1 nmol of fucose is demonstrated. Analytical methods and results are presented for mannose, fucose, and galactose in serum glycoproteins for both normal women and those with metastatic breast cancer. We briefly discuss the possibility of separating and analyzing for the three neutral carbohydrates in serum glycoproteins in 4 h by isocratic (constant eluent concentration) elution from a chromatographic column. (+info)