Measurement of enzyme kinetics using a continuous-flow microfluidic system.
AuthorsSeong GH, Heo J, Crooks RM
JournalAnal Chem
PubMed ID12964765
'This paper describes a microanalytical method for determining enzyme kinetics using a continuous-flow microfluidic system. The analysis is carried out by immobilizing the enzyme on microbeads, packing the microbeads into a chip-based microreactor (volume approximately 1.0 nL), and flowing the substrate over the packed bed. Data were analyzed using the ... More
Crystallization of beta-galactosidase does not reduce the range of activity of individual molecules.
AuthorsShoemaker GK, Juers DH, Coombs JM, Matthews BW, Craig DB
JournalBiochemistry
PubMed ID12578385
'By use of a capillary electrophoresis-based procedure, it is possible to measure the activity of individual molecules of beta-galactosidase. Molecules from the crystallized enzyme as well as the original enzyme preparation used to grow the crystals both displayed a range of activity of 20-fold or greater. beta-Galactosidase molecules obtained from ... More
Stochastic inhibitor release and binding from single-enzyme molecules.
AuthorsGorris HH, Rissin DM, Walt DR,
JournalProc Natl Acad Sci U S A
PubMed ID17965235
'Inhibition kinetics of single-beta-galactosidase molecules with the slow-binding inhibitor d-galactal have been characterized by segregating individual enzyme molecules in an array of 50,000 ultra small reaction containers and observing substrate turnover changes with fluorescence microscopy. Inhibited and active states of beta-galactosidase could be clearly distinguished, and the large array size ... More
'Many areas of research today are based on enzymatic assays most of which are still performed as enzyme-linked immunosorbent assays in microtiter plates. The demand for highly parallel screening of thousands of samples eventually led to a miniaturization and automation of these assays. However, the final transfer of enzymatic assays ... More
Microchip device for performing enzyme assays.
AuthorsHadd AG, Raymond DE, Halliwell JW, Jacobson SC, Ramsey JM
JournalAnal Chem
PubMed ID9286159
'An automated enzyme assay was performed within a microfabricated channel network. Precise concentrations of substrate, enzyme, and inhibitor were mixed in nanoliter volumes using electrokinetic flow. Reagent dilution and mixing were controlled by regulating the applied potential at the terminus of each channel, using voltages derived from an equivalent circuit ... More
Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited.
AuthorsEnglish BP, Min W, van Oijen AM, Lee KT, Luo G, Sun H, Cherayil BJ, Kou SC, Xie XS
JournalNat Chem Biol
PubMed ID16415859
'Enzymes are biological catalysts vital to life processes and have attracted century-long investigation. The classic Michaelis-Menten mechanism provides a highly satisfactory description of catalytic activities for large ensembles of enzyme molecules. Here we tested the Michaelis-Menten equation at the single-molecule level. We monitored long time traces of enzymatic turnovers for ... More
Expression of beta-galactosidase under the control of the human c-myc promoter in transgenic mice is inhibited by mithramycin.
AuthorsJones DE, Cui DM, Miller DM
JournalOncogene
PubMed ID7784080
'In order to assess the functional contribution of the human c-myc promoter region in the expression of the c-myc gene, transgenic mouse lines containing a bacterial lac Z gene encoding beta-galactosidase under the control of the human c-myc protooncogene promoter were generated. Transgenic mouse embryos heterozygous for the human c-myc ... More
Cell lysis and protein extraction in a microfluidic device with detection by a fluorogenic enzyme assay.
AuthorsSchilling EA, Kamholz AE, Yager P
JournalAnal Chem
PubMed ID11985310
'A critical requirement for achieving a micro total analytical system for the analysis of cells and their constituent proteins is to integrate the lysis and fractionation steps on-chip. Here, an experimental microfluidic system integrating the lysis of bacterial cells and the extraction of a large intracellular enzyme, beta-galactosidase, is demonstrated. ... More
Enzyme immunoassay techniques. An overview.
AuthorsPorstmann T, Kiessig ST
JournalJ Immunol Methods
PubMed ID1613258
'In spite of the great variety of enzyme immunoassays (EIA) they can be classified into two groups 'analyte-observed' and 'reagent-observed' assays, depending on their reaction principle. The latter are favored by use of monoclonal antibodies and are characterized by a greater sensitivity, a larger measuring range, a lower susceptibility to ... More
Digital concentration readout of single enzyme molecules using femtoliter arrays and Poisson statistics.
AuthorsRissin DM, Walt DR
JournalNano Lett
PubMed ID16522055
'Methods for accurately quantifying the concentration of a particular analyte in solution are all based on ensemble responses in which many analyte molecules give rise to the measured signal. In this paper, single molecules of beta-galactosidase were monitored using a 1 mm diameter fiber optic bundle with 2.4 x 10(5) ... More
Dispersive analysis of turnover rates of a CST reactor by flow-through microfluorometry under conditions of growth.
AuthorsSernetz M, Willems H, Keiner K
JournalAnn N Y Acad Sci
PubMed ID2127518
The GUS reporter gene system.
AuthorsJefferson RA
JournalNature
PubMed ID2689886
The GUS reporter gene system is already a powerful tool for the assessment of gene activity in transgenic plants. Further developments may lead to routine in vivo analysis and fusion genetics. ... More
beta-galactosidase assay using capillary electrophoresis laser-induced fluorescence detection and resorufin-beta-D-galactopyranoside as substrate.
AuthorsEggertson MJ, Craig DB
JournalBiomed Chromatogr
PubMed ID10611604
beta-Galactosidase was incubated for 60 min with the fluorogenic substrate resorufin-beta-D-galactopyranoside, which is converted by the action of the enzyme into resorufin and galactose. A 160 pL aliquot of reaction mixture was analyzed by capillary electrophoresis utilizing laser-induced fluorescence detection. Based on the detection of the resorufin formed, the limit ... More
A single-cell assay of beta-galactosidase activity in Saccharomyces cerevisiae.
AuthorsWittrup KD, Bailey JE
JournalCytometry
PubMed ID3135986
A novel assay of single-cell exogenous beta-galactosidase activity in Saccharomyces cerevisiae has been developed. Intracellular fluorescence due to the hydrolysis of resorufin-beta-D-galactopyranoside attains a steady state between production of resorufin and its subsequent leakage from the cell. The cells are permeabilized with Triton X-100, and the assay is performed at ... More