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Reaction Volume (reaction + volume)
Selected AbstractsEfficient and scalable method for scaling up cell free protein synthesis in batch modeBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2005Alexei M. Voloshin A novel method for general cell free system scale-up in batch mode was applied to expression of E. coli chloramphenicol acetyl transferase (CAT) and a GMCSF-scFv fusion protein being developed as a B-cell lymphoma vaccine candidate (GLH). Performance of two different E. coli based cell-free systems was evaluated using the new scale-up approach. Reaction volumes from 15 to 500 µL were tested for both products and both reaction systems. In each case, the new scale-up method preserved total, soluble, and active volumetric yields of GLH and CAT at every reaction volume. At the 500 µL reaction volume, the PANOx SP system produced 560,±,36 µg/mL of active CAT and 99,±,10 µg/mL of active GLH protein using the new thin film approach whereas 500 µL test tube reactions produced 250,±,42 µg/mL and 72,±,7 µg/mL of active CAT and GLH respectively. Similarly, 500 µL cell-free synthesis reactions with the Cytomim system produced 481,±,38 µg/mL of active CAT and 109,±,15 µg/mL active GLH respectively in thin films compared to 29,±,7 µg/mL of active CAT and 5,±,2 µg/mL of active GLH protein in 500 µL test tube reactions. The new thin film approach improves oxygen supply for the Cytomim system, and increases the availability of hydrophobic surfaces. Analysis suggests that these surfaces provide significant benefit for protein expression and folding. We believe that this approach provides a general reaction scale-up technology that will be suitable for any protein target, cell free system, and reaction volume. © 2005 Wiley Periodicals, Inc. [source] Simply and reliably integrating micro heaters/sensors in a monolithic PCR-CE microfluidic genetic analysis systemELECTROPHORESIS, Issue 8 2009Runtao Zhong Abstract A novel fabrication process was presented to construct a monolithic integrated PCR-CE microfluidic DNA analysis system as a step toward building a total genetic analysis microsystem. Microfabricated Titanium/Platinum (Ti/Pt) heaters and resistance temperature detectors (RTDs) were integrated on the backside of a bonded glass chip to provide good thermal transfer and precise temperature detection for the drilled PCR-wells. This heater/RTD integration procedure was simple and reliable, and the resulting metal layer can be easily renewed when the Ti/Pt layer was damaged in later use or novel heater/RTD design was desired. A straightforward "RTD-calibration" method was employed to optimize the chip-based thermal cycling conditions. This method was convenient and rapid, comparing with a conventional RTD-calibration/temperature adjustment method. The highest ramping rates of 14°C/s for heating and 5°C/s for cooling in a 3-,L reaction volume allow 30 complete PCR cycles in about 33,min. After effectively passivating the PCR-well surface, successful ,-phage DNA amplifications were achieved using a two- or three-temperature cycling protocol. The functionality and performance of the integrated microsystem were demonstrated by successful amplification and subsequent on-line separation/sizing of ,-phage DNA. A rapid assay for Hepatitis B virus, one of the major human pathogens, was performed in less than 45,min, demonstrating that the developed PCR-CE microsystem was capable of performing automatic and high-speed genetic analysis. [source] Electrophoretically mediated reaction of glycosidases at a nanoliter scaleELECTROPHORESIS, Issue 6 2003Yoshimi Kanie Abstract We have investigated electrophoretically mediated microanalysis (EMMA) for the assay of a native glyco-enzyme. As a representative of this class of enzyme, ,-glucosidase was selected, and the reaction was analyzed. Our EMMA was based on the plug-plug interaction of enzyme and substrate plugs, which is essential to reduce quantities of materials. Furthermore, we have addressed the problem of incompatibility of the enzymatic reaction and separation of the reactants. As a result, EMMA of native glycosidase was achieved with a reaction volume of ,,20 nL and the Michaelis constant was estimated according to the Lineweaver-Burk plot. The current method may have advantages over traditional assay methods, especially in terms of the amount of enzyme (ng order) and substrate (pmol order) required for a reaction*. [source] Regioselective C-6 Hydrolysis of Methyl O -Benzoyl-pyranosides Catalysed by Candida Rugosa LipaseEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 10 2009Aslan Esmurziev Abstract Hydrolysis of six methyl O -benzoyl-pyranosides has been investigated using Candida rugosa lipase in dioxane/buffer mixtures. The lipase catalysed the hydrolysis of all substrates in a regiospecific manner at C-6. The rate of reaction was dependent on pyranoside structure, reaction temperature and scale, dioxane concentration and agitation speed. Starting from their C-6 O -benzoyl precursors, the methyl 2,3,4-tri- O -benzoyl-pyranosides of ,- D -galactose, ,- D -galactose, ,- D -glucose, and methyl 2,3-di- O -benzoyl-,- D -galactopyranoside could be isolated in 85,96,% yield. In hydrolysis of methyl 2,3,4,6-tetra- O -benzoyl-,- D -glucopyranoside and methyl 2,3,4,6-tetra- O -benzoyl-,- D -galactopyranoside substrate inhibition were observed, which in part could be overcome by increasing the reaction volume. Methyl 2,3,4,6-tetra- O -benzoyl-,- D -glucopyranoside and methyl 2,3,4,6-tetra- O -benzoyl-,- D -mannopyranoside were poor substrates for Candida rugosa lipase and low degree of conversion towards products were obtained under all conditions. No acyl migration was detected in any of the products.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) [source] Properties and application of poly(methacrylic acid- co -dodecyl methacrylate- cl - N,N -methylene bisacrylamide) hydrogel immobilized Bacillus cereus MTCC 8372 lipase for the synthesis of geranyl acetateJOURNAL OF APPLIED POLYMER SCIENCE, Issue 2 2008Madan Lal Verma Abstract A range of fatty acid esters is now being produced commercially with immobilized microbial lipases (glycerol ester hydrolases; EC) in nonaqueous solvents. In this study, a synthetic hydrogel was prepared by the copolymerization of methacrylic acid and dodecyl methacrylate in the presence of a crosslinker, N,N -methylene bisacrylamide. A purified alkaline thermotolerant bacterial lipase from Bacilluscereus MTCC 8372 was immobilized on a poly(methacrylic acid- co -dodecyl methacrylate- cl - N,N -methylene bisacrylamide) hydrogel by an adsorption method. The hydrogel showed a 95% binding efficiency for the lipase. The bound lipase was evaluated for its hydrolytic potential toward various p -nitrophenyl acyl esters with various C chain lengths. The bound lipase showed optimal hydrolytic activity toward p -nitrophenyl palmitate at a pH of 8.5 and a temperature of 55°C. The hydrolytic activity of the hydrogel-bound lipase was enhanced by Hg2+, Fe3+, and NH ions at a concentration of 1 mM. The hydrogel-bound lipase was used to synthesize geranyl acetate from geraniol and acetic acid in n -heptane. The optimization of the reaction conditions, such as catalyst loading, effect of substrate concentration, solvent (n -pentane, n -hexane, n -heptane, n -octane, and n -nonane), reaction time, temperature, molecular sieve (3 Å × 1.5 mm) and scale up (at 50-mL level), was studied. The immobilized lipase (25 mg/mL) was used to perform an esterification in n -alkane(s) that resulted in the synthesis of approximately 82.8 mM geranyl acetate at 55°C in n -heptane under continuous shaking (160 rpm) after 15 h when geraniol and acetic acid were used in a ratio of 100 : 100 mM. The addition of a molecular sieve (3 Å × 1.5 mm) to the reaction system at a concentration of 40 mg/mL in reaction volume (2 mL) resulted in an increase in the conversion of reactants into geranyl acetate (90.0 mM). During the repetitive esterification under optimum conditions, the hydrogel-bound lipase produced ester (37.0 mM) after the eighth cycle of reuse. When the reaction volume was scaled up to 50 mL, the ester synthesized was 58.7 mM under optimized conditions. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008 [source] False metamorphic events inferred from misinterpretation of microstructural evidence and P,T dataJOURNAL OF METAMORPHIC GEOLOGY, Issue 4 2008R. H. VERNON Abstract Geometrical relationships involving inclusions and partial inclusions in metamorphic microstructures can be inadequate for inferring an order of crystallization and hence a metamorphic reaction. Unique spatial and/or chemical relationships need to be defined for mineral inclusions, in the context of a reference paragenesis, commonly the matrix assemblage. Corona microstructures are reliable indicators of metamorphic reactions, but require considerable care when used to infer reactions or changes in P,T conditions, owing to kinetic problems, as well as to changes in the effective reaction volume during changes across relatively broad P,T stability fields of assemblages. Mineral equilibria models, most commonly implemented through P,T pseudosections, may allow the order in which different minerals become stable along a given P,T path to be inferred. However, the order in which two minerals become stable may be different from the order in which two grains of these minerals nucleate. Furthermore, such diagrams cannot make predictions about which minerals will form porphyroblasts and which minerals will form inclusions in porphyroblasts. An evaluation of three examples from the Australian Proterozoic shows that modelling, in combination with inclusion-host relationships, is a powerful tool for understanding the metamorphic evolution of a rock, but involves considerable uncertainty. [source] Particle-induced myeloperoxidase release in serially diluted whole blood quantifies the number and the phagocytic activity of blood neutrophils and opsonization capacity of plasmaLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 3 2006Esa-Matti Eino Lilius Abstract Luminol-amplified chemiluminescence (CL) from phagocytes has previously been shown to be almost completely dependent on the release of myeloperoxidase (MPO) from azurophilic granules. We measured the luminol-amplified chemiluminescence response (WBCL) by using serially diluted whole blood. In these experiments, non-opsonized and serum-opsonized zymosan (NWBCL and OWBCL, respectively) were used concurrently as phagocytosable particles. We found two whole-blood dilution ranges with clinical significance: first, <0.04% of whole blood in the reaction volume, where MPO released by the zymosan-activated leukocyte population came almost totally from neutrophils and the OWBCL response could be exploited as a measure of a neutrophil count in a given blood specimen, despite the pathophysiological state of the host. In contrast, the NWBCL response was two-fold in blood samples from bacterial infection patients compared to those of controls and patients with viral infection, suggesting the use of NWBCL for the differential diagnosis of bacterial infections from viral infections; second, 0.16,1.2% of whole blood in the reaction volume, where the opsonization capacity of plasma (OC50) can be determined. We also found that at whole blood content >0.04%, erythrocytes quickly start to absorb chemiluminescence light, and that at whole blood content >1.2%, plasma proteins, most probably albumin and fibrinogen, start to inhibit MPO release. Copyright © 2006 John Wiley & Sons, Ltd. [source] Development of a PCR test to detect the downy mildew causal agent Plasmopara halstedii in sunflower seedsPLANT PATHOLOGY, Issue 2 2007R. Ioos Plasmopara halstedii, the causal agent of downy mildew of sunflower, is an obligate parasite but viable sporangia and oospores of the pathogen may be found in a quiescent state in seeds of sunflower and therefore may be transported with sunflower seeds in international commercial exchanges. In order to prevent the spread of this pathogen, especially the introduction of potentially new races, an efficient method to analyse sunflower seed samples is required. In this study, a P. halstedii -specific polymerase chain reaction (PCR) test was developed based on the ribosomal large sub unit (LSU) DNA. The forward (PHAL-F) and reverse (PHAL-R) PCR primers were designed from two polymorphic regions of LSU. After screening 22 isolates of P. halstedii corresponding to different races and countries and 32 other oomycete, deuteromycete and ascomycete isolates, the PHAL-F/R primers amplified only a single PCR band of c. 310 bp from P. halstedii. The PHAL-F/R PCR test could detect as little as 3 pg of P. halstedii genomic DNA per 20 µL reaction volume and enabled the direct detection of P. halstedii in 35 g sunflower seed samples without the need for any prior biological baiting step. An internal amplification control (IAC) was developed to help discriminate against false negative samples due to the potential presence of inhibitory compounds in DNA extracts. The test was successfully used on samples of naturally contaminated seeds. These new molecular tools should be of great interest for quarantine seed testing purposes. [source] Technical note: PCR analysis of minimum target amount of ancient DNAAMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY, Issue 2 2010Daniela Woide Abstract The study of ancient DNA plays an important role in archaeological and palaeontological research as well as in pathology and forensics. Here, we present a new tool for ancient DNA analysis, which overcomes contamination problems, DNA degradation, and the negative effects of PCR inhibitors while reducing the amount of starting target material in the picogram range. Ancient bone samples from four Egyptian mummies were examined by combining laser microdissection, conventional DNA extraction, and low-volume PCR. Initially, several bone particles (osteons) in the micrometer range were extracted by laser microdissection. Subsequently, ancient DNA amplification was performed to verify our extraction method. Amelogenin and ,-actin gene specific fragments were amplified via low-volume PCR in a total reaction volume of 1 ,l. Results of microdissected mummy DNA samples were compared to mummy DNA, which was extracted using a standard DNA extraction method based on pulverization of bone material. Our results highlight the combination of laser microdissection and low-volume PCR as a promising new technique in ancient DNA analysis. Am J Phys Anthropol, 2010. © 2010 Wiley-Liss, Inc. [source] Efficient and scalable method for scaling up cell free protein synthesis in batch modeBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2005Alexei M. Voloshin A novel method for general cell free system scale-up in batch mode was applied to expression of E. coli chloramphenicol acetyl transferase (CAT) and a GMCSF-scFv fusion protein being developed as a B-cell lymphoma vaccine candidate (GLH). Performance of two different E. coli based cell-free systems was evaluated using the new scale-up approach. Reaction volumes from 15 to 500 µL were tested for both products and both reaction systems. In each case, the new scale-up method preserved total, soluble, and active volumetric yields of GLH and CAT at every reaction volume. At the 500 µL reaction volume, the PANOx SP system produced 560,±,36 µg/mL of active CAT and 99,±,10 µg/mL of active GLH protein using the new thin film approach whereas 500 µL test tube reactions produced 250,±,42 µg/mL and 72,±,7 µg/mL of active CAT and GLH respectively. Similarly, 500 µL cell-free synthesis reactions with the Cytomim system produced 481,±,38 µg/mL of active CAT and 109,±,15 µg/mL active GLH respectively in thin films compared to 29,±,7 µg/mL of active CAT and 5,±,2 µg/mL of active GLH protein in 500 µL test tube reactions. The new thin film approach improves oxygen supply for the Cytomim system, and increases the availability of hydrophobic surfaces. Analysis suggests that these surfaces provide significant benefit for protein expression and folding. We believe that this approach provides a general reaction scale-up technology that will be suitable for any protein target, cell free system, and reaction volume. © 2005 Wiley Periodicals, Inc. [source] Multienzyme catalysis in microfluidic biochipsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2003Moo-Yeal Lee Abstract The attachment of enzymes to glass microfluidic channels has been achieved using a highly reactive poly(maleic anhydride- alt -,-olefin) (PMA)-based coating that is supplied to the microchannel in a toluene solution. The PMA reacts with 3-aminopropyltriethoxysilane groups linked to the glass surface to form a matrix that enables additional maleic anhydride groups to react with free amino groups on enzymes to give a mixed covalent,noncovalent immobilization support. Using a simple T-channel microfluidic design, with reaction channel dimensions of 200 ,m wide (at the center), 15 ,m deep, and 30 mm long giving a reaction volume of 90 nL, soybean peroxidase (SBP) was attached at an amount up to 0.6 ,g/channel. SBP-catalyzed oxidation of p -cresol was performed in aqueous buffer (with 20% [v/v], dimethylformamide) containing H2O2, with microfluidic transport enabled by electroosmotic flow (EOF). Michaelis,Menten kinetics were obtained with Km and Vmax values of 0.98 mM and 0.21 ,mol H2O2 converted/mg SBP per minute, respectively. These values are nearly identical to nonimmobilized SBP kinetics in aqueous,DMF solutions in 20-,L volumes in 384-well plates and 5-mL reaction volumes in 20-mL scintillation vials. These results indicate that SBP displays intrinsically native activity even in the immobilized form at the microscale, and further attests to the mild immobilization conditions afforded by PMA. Bienzymic and trienzymic reactions were also performed in the microfluidic biochip. Specifically, a combined Candida antarctica lipase B,SBP bienzymic system was used to convert tolyl acetate into poly(p -cresol), and an invertase,glucose oxidase SBP trienzymic system was used to take sucrose and generate H2O2 for SBP-catalyzed synthesis of poly(p -cresol). © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 20,28, 2003. [source] Cell-Free Protein Synthesis System Prepared from Insect Cells by Freeze-ThawingBIOTECHNOLOGY PROGRESS, Issue 6 2006Toru Ezure We established a novel cell-free protein synthesis system derived from Trichoplusia ni (HighFive) insect cells by a simple extraction method. Luciferase and ,-galactosidase were synthesized in this system with active forms. We analyzed and optimized (1) the preparation method of the insect cell extract, (2) the concentration of the reaction components, and (3) the 5,-untranslated region (5,-UTR) of mRNA. The extract was prepared by freeze-thawing insect cells suspended in the extraction buffer. This preparation method was a simple and superior method compared with the conventional method using a Dounce homogenizer. Furthermore, protein synthesis efficiency was improved by the addition of 20% (v/v) glycerol to the extraction buffer. Concentrations of the reaction components were optimized to increase protein synthesis efficiency. Moreover, mRNAs containing 5,-UTRs derived from baculovirus polyhedrin genes showed high protein synthesis activity. Especially, the leader composition of the Ectropis obliqua nucleopolyhedrovirus polyhedrin gene showed the highest enhancement activity among the six 5,-UTRs tested. As a result, in a batch reaction approximately 71 ,g of luciferase was synthesized per milliliter of reaction volume at 25 °C for 6 h. Moreover, this method for the establishment of a cell-free system was applied also to Spodoptera frugiperda 21 (Sf21) insect cells. After optimizing the concentrations of the reaction components and the 5,-UTR of mRNA, approximately 45 ,g/mL of luciferase was synthesized in an Sf21 cell-free system at 25 °C for 3 h. These productivities were sufficient to perform gene expression analyses. Thus, these cell-free systems may be a useful tool for simple synthesis in post-genomic studies as a novel protein production method. [source] Bioconversion of Linoleic Acid into Conjugated Linoleic Acid by Immobilized Lactobacillus reuteriBIOTECHNOLOGY PROGRESS, Issue 3 2003Sun-Ok Lee Lactobacillus reuteri was immobilized on silica gel to evaluate the bioconversion of linoleic acid (LA) into conjugated linoleic acid (CLA), consisting of cis -9,trans -11 and trans -10,cis -12 isomers. The amount of cell to carrier, the reaction time, and the substrate concentration, pH, and temperature for CLA production were optimized at 10 mg of cells/(g of carrier), 1 h, 500 mg/L LA, 10.5, and 55 °C, respectively. In the presence of 1.0 mM Cu2+, CLA production increased by 110%. Under the optimal conditions, the immobilized cells produced 175 mg/L CLA from 500 mg/L LA for 1 h with a productivity of 175 mg/(L·h) and accumulated 5.5 times more CLA than that obtained from bioconversion by free washed cells. The CLA-producing ability of reused cells was investigated over five reuse reactions and was maximal at pH 7.5, 25 °C, and 1.0 mM Cu2+. The total amount of CLA by the combined five reuse reactions was 344 mg of CLA/L reaction volume. This was 8.6 times higher than the amount obtained from reuse reactions by free washed cells. [source] Guidance on Safety/Health for Process Intensification including MS Design; Part I: Reaction HazardsCHEMICAL ENGINEERING & TECHNOLOGY (CET), Issue 11 2009O. Klais Abstract The implementation of process intensification by multiscale equipment will have a profound impact on the way chemicals are produced. The shift to higher space-time yields, higher temperatures, and a confined reaction volume comprises new risks, such as runaway reactions, decomposition, and incomplete conversion of reactants. Simplified spreadsheet calculations enable an estimation of the expected temperature profiles, conversion rates, and consequences of potential malfunction based on the reaction kinetics. The analysis illustrates that the range of optimal reaction conditions is almost congruent with the danger of an uncontrolled reaction. The risk of a spontaneous reaction with hot spots can be presumed if strong exothermic reactions are carried out in micro-designed reactors. At worst, decomposition follows the runaway reaction with the release of noncondensable gases. Calculations prove that a microreactor is not at risk in terms of overpressure as long as at least one end of the reactor is not blocked. [source] The Influence of Differences Between Microchannels on Micro Reactor PerformanceCHEMICAL ENGINEERING & TECHNOLOGY (CET), Issue 3 2005E. R. Delsman Abstract Microstructured reactors most often contain a large number of micrometer-sized, parallel channels, instead of a large undivided reaction volume. Individual microchannels behave as plug-flow reactors without significant axial dispersion and with excellent heat and mass transfer properties. However, since the reaction takes place in a large number of parallel channels, it is important that all channels provide equal residence time and amount of catalyst volume. These issues depend not only on the flow distributor design, but also, for example, on the manufacturing tolerances. Correlations are derived to express the conversion of a multichannel microreactor explicitly as a function of the variance of a number of reactor parameters, viz. the channel flow rate, the channel diameter, the amount of catalyst in a channel, and the channel temperature. It is shown that the influence of flow maldistribution on the overall reactor conversion is relatively small, while the influences of variations in the channel diameter and the amount of catalyst coating are more pronounced. The model outcomes are also compared to experimental results of two microreactors with different catalyst distributions, which show that the presented method is able to provide a quick, though rough estimation of the influence of differences between channels on microreactor performance. [source] Nano-sized bacterial magnetic particles displaying pyruvate phosphate dikinase for pyrosequencingBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Tomoko Yoshino Abstract There is a high demand for inexpensive and high-throughput DNA sequencing technologies in molecular biology and applied biosciences. In this study, novel nano-sized magnetic particles displaying enzymes for pyrosequencing, a rather novel bioluminometric DNA sequencing method based on the sequencing-by-synthesis principle by employing a cascade of several enzymatic reactions, was developed. A highly thermostable enzyme, pyruvate phosphate dikinase (PPDK) which converts PPi to ATP was successfully expressed onto bacterial magnetic particles (BacMPs) using a novel protein display system of Magnetospirillum magneticum AMB-1. The enzymatic stability of BacMPs displaying PPDK (PPDK-BacMPs) to pH and temperature was evaluated and its broad range of properties was shown. Subsequently, PPDK-BacMPs were applied in pyrosequencing and a target oligonucleotide was successfully sequenced. The PPDK enzyme displayed on BacMPs was shown to be recyclable in each sequence reaction as they can be manipulated by magnetic force. It was concluded that nano-sized PPDK-BacMPs are useful for the scale down of pyrosequencing reaction volumes, thus, permitting high-throughput. The recycling of enzymes was also shown to be promising and applicable for the development of an inexpensive DNA sequencing at a low running cost. Biotechnol. Bioeng. 2009;103: 130,137. © 2008 Wiley Periodicals, Inc. [source] Multienzyme catalysis in microfluidic biochipsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2003Moo-Yeal Lee Abstract The attachment of enzymes to glass microfluidic channels has been achieved using a highly reactive poly(maleic anhydride- alt -,-olefin) (PMA)-based coating that is supplied to the microchannel in a toluene solution. The PMA reacts with 3-aminopropyltriethoxysilane groups linked to the glass surface to form a matrix that enables additional maleic anhydride groups to react with free amino groups on enzymes to give a mixed covalent,noncovalent immobilization support. Using a simple T-channel microfluidic design, with reaction channel dimensions of 200 ,m wide (at the center), 15 ,m deep, and 30 mm long giving a reaction volume of 90 nL, soybean peroxidase (SBP) was attached at an amount up to 0.6 ,g/channel. SBP-catalyzed oxidation of p -cresol was performed in aqueous buffer (with 20% [v/v], dimethylformamide) containing H2O2, with microfluidic transport enabled by electroosmotic flow (EOF). Michaelis,Menten kinetics were obtained with Km and Vmax values of 0.98 mM and 0.21 ,mol H2O2 converted/mg SBP per minute, respectively. These values are nearly identical to nonimmobilized SBP kinetics in aqueous,DMF solutions in 20-,L volumes in 384-well plates and 5-mL reaction volumes in 20-mL scintillation vials. These results indicate that SBP displays intrinsically native activity even in the immobilized form at the microscale, and further attests to the mild immobilization conditions afforded by PMA. Bienzymic and trienzymic reactions were also performed in the microfluidic biochip. Specifically, a combined Candida antarctica lipase B,SBP bienzymic system was used to convert tolyl acetate into poly(p -cresol), and an invertase,glucose oxidase SBP trienzymic system was used to take sucrose and generate H2O2 for SBP-catalyzed synthesis of poly(p -cresol). © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 20,28, 2003. [source] An ESI-MS/MS Method for Screening of Small-Molecule Mixtures against Glycogen Synthase Kinase-3, (GSK-3,)CHEMBIOCHEM, Issue 7 2008Ivan Partserniak Abstract Glycogen synthase kinase-3, (GSK-3,) is involved in the hyperphosphorylation of previously phosphorylated (primed) substrates, and is currently assayed using an approach based on the incorporation of ,- 32P-radiolabelled isotopes into substrate peptides. The requirement to detect hyperphosphorylation of a primed substrate poses a particular challenge for development of a high-throughput screening assay, as many current kinase assays are designed to produce a signal in the presence of any phosphorylation site, and thus are only suitable for ,-unphosphorylated substrates. Herein, we have developed an electrospray-ionization tandem mass spectrometry (ESI-MS/MS) assay to allow for direct detection of a hyperphosphorylated product which is formed in a solution reaction involving a primed peptide substrate (GSM peptide) and GSK-3,. Optimum reaction conditions (level of Mg2+, buffer type, ionic strength, pH, enzyme concentration, and reaction time) were established to both maintain the activity of GSK-3, and allow for substrate and product quantification through ESI/MS/MS. We show that the MS-based assay allows for rapid determination of GSK-3, activity from reaction volumes of ,40 ,L and that it can be used to assess IC50 values and the site of action of known inhibitors. It also can be used for automated screening of small-molecules mixtures to identify inhibitors of GSK-3,. [source] | |||||||||||||