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Chemistry
Chemical Measurements: Absorbance
Chemical Measurements: Luminescence
chemical measurements: absorbance
cholesterol & triglycerides
Ultrasensitive enzymatic cholesterol and triglyceride profiles of density gradient lipoprotein fractions
8th International Symposium on Atherosclerosis, Roma, International Atherosclerosis Society (eds), CIC Edizioni Internazionali, (Roma). (1988).
John D. Belcher and Jack O. Egan.
University of Minnesota, Minneapolis, Minnesota USA.
Summary. Microassays were developed for the measurement of total cholesterol (TC) and triglyceride (TG) by enzymatic methods. The assays are linear between 1 and 100 mg/dL with a sensitivity of 1 mg/dL for both TC and TG. The assay uses 30 µL of sample or standard which is added to each well followed by 150 µL of either TC or TG enzymatic reagent. The microplates were incubated at room temperature for 15 minutes and read in a VMax microplate reader at 490 nm with a 650 nm reference. The mean accuracy of the assay was -0.5% and -0.7% for TC and TG, respectively, and the within- and between-assay coefficients of variation were below 3%.
citrate & glucose consumption
Mechanism of citrate metabolism in Lactococcus lactis: resistance against lactate toxicity at low pH
J. Bacteriol. 181: 1451–57 (1999).
Christian Magni1,2, Diego de Mendoza2, Wil N. Konings1, and Juke S. Lolkema1.
1 Department of Microbiology, Groningen Biotechnology and Biomolecular Sciences Institute, University of Groningen, 9751 NN Haren, The Netherlands.
2 Programa Multidisciplinario de Biología Experimental (ProMUBIE-CONICET) and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmaceuticas, Universidad Nacional de Rosario, 2000 Rosario, Argentina.
Methods: measurement of citrate and glucose consumption rates. Cells were harvested and resuspended to an OD660 of 6 in 50 mM potassium phosphate buffer (pH 5.5). Citrate utilization was initiated by the addition of 500 µL of cells to 1,500 µL of buffer containing 2 mM citrate. When indicated, glucose and lactate were included at concentrations of 0.5 and 2 mM, respectively. Glucose utilization was initiated by addition of 50 µL of cells to 1,950 µL of buffer containing 0.5 mM glucose and 2 mM citrate when indicated. At the indicated times, 250 µL of the cell suspension was centrifuged for 15 seconds in an Eppendorf tabletop centrifuge operating at maximal speed, and a sample of the supernatant was stored in liquid nitrogen until analysis. The concentrations of citrate, pyruvate, and glucose in the supernatants were measured by commercially available enzyme kits for citrate and D-glucose (Boehringer). The protocol for the measurement of citrate was modified slightly to allow the measurement of pyruvate (and oxaloacetate) at the same time, as described before. Both protocols were modified for use in 96-well plates as follows. For the citrate assay, 100 µL of the buffer provided by the manufacturer was mixed with 50 µL of water, after which the A340 was measured. Subsequently, 50 µL of the supernatant was added and the OD was read again. The difference between the two readings is a measure of the pyruvate (or oxaloacetate) concentration in the sample. After addition of 1 µL of the citrate lyase solution, the absorbance was read again, which provides the data for the calculation of the citrate concentration as indicated in the protocol. For glucose determination, the volumes in the manufacturer’s protocol were scaled down to give a total volume of 200 µL. The ODs were read by using a SpectraMax 340 microplate spectrophotometer (Molecular Devices Corp.)
heavy metals
Cu(II) potentiation of Alzheimer A neurotoxicity: correlation with cell-free hydrogen peroxide production and metal reduction
J. Biol. Chem. 52: 37111–16 (1999).
Xudong Huang, Math P. Cuajungco, Craig S. Atwood, Mariana A. Hartshorn, Joel D. A. Tyndall, Graeme R. Hanson, Karen C. Stokes, Michael Leopold, Gerd Multhaup, Lee E. Goldstein, Richard C. Scarpa, Aleister J. Saunders, James Lim, Robert D. Moir, Charles Glabe, Edmond F. Bowden, Colin L. Masters, David P. Fairlie, Rudolph E. Tanzi, and Ashley I. Bush.
Methods: metal reduction assays. Assays were performed using a 96-well microtiter plate (Costar), based upon a modification of established protocols. Polypeptides (10 µM) or vitamin C (10 µM), Cu(II)-glycine and Cu(I) indicator (250 µM), either bathocuproine disulfonic acid (BC) or bicinchoninic acid (BCA, 4,4´-dicarboxy-2,2´-biquinoline), were coincubated in PBS at 37 °C. Absorbance was measured using a plate reader (SpectraMax Plus, Molecular Devices Corp.) In control samples, both metal ion and indicator were present to determine the background buffer signal. Absorbance of metal ion and peptide present in the absence of indicator were taken to estimate the contribution of light scattering due to turbidity. The net absorbance (A) was obtained by deducting the absorbance from these controls from the absorbance generated by the peptide and metal in the presence of the indicator. Cu(I) concentrations (µM) were calculated as A x 106/M, where M is the known molar absorption coefficient (M-1 cm-1). For Cu(I)-BC, M = 12,250 at 483 nm; and for Cu(I)-BCA, M = 7700 at 562 nm.
Dramatic aggregation of Alzheimer A by Cu(II) is induced by conditions representing physiological acidosis
J. Biol. Chem. 273: 12817–26 (1998).
Craig S. Atwood1, Robert D. Moir2, Xudong Huang1, Richard C. Scarpa1, N. Michael E. Bacarra1, Donna M. Romano2, Mariana A. Hartshorn1, Rudolph E. Tanzi2, and Ashley I. Bush3.
1 Department of Psychiatry and Genetics and Aging Unit.
2 Department of Neurology and Genetics and Aging Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114.
Methods: Cu2+-induced spectral changes in amyloid protein. Cu2+-induced spectral changes in A were monitored by incubating A1-40 or A1-42 (5 µM) in 20 mM ammonium acetate buffer, 150 mM NaCl (pH 7.4), with and without various concentrations of Cu2+ for 5 minutes prior to loading onto a quartz microtiter plate. Absorbance was scanned between 200 and 800 nm on a SpectraMax Plus spectrophotometer (Molecular Devices Corp.) Incubation of Cu2+ with A induced a change in the absorbance profile of the peptide, which was maximal at 208 nm. However, subsequent analyses were performed at 214 nm where the spectral shift was still large but the background signal was lower.
hydrogen peroxide
Cu(II) potentiation of Alzheimer A neurotoxicity: correlation with cell-free hydrogen peroxide production and metal reduction
J. Biol. Chem. 274: 37111–16 (1999).
Xudong Huang1, Math P. Cuajungco1, Craig S. Atwood1, Mariana A. Hartshorn1, Joel D. A. Tyndall3, Graeme R. Hanson4, Karen C. Stokes5, Michael Leopold5, Gerd Multhaup6, Lee E. Goldstein1, Richard C. Scarpa1, Aleister J. Saunders1, James Lim1, Robert D. Moir7, Charles Glabe9, Edmond F. Bowden5, Colin L. Masters10, David P. Fairlie3, Rudolph E. Tanzi7, and Ashley I. Bush11.
1 Laboratory for Oxidation Biology, Genetics and Aging Unit, and Department of Psychiatry, Harvard Medical School, Massachusetts General Hospital, Charlestown, Massachusetts 02129.
3 Centre for Drug Design and Development.
4 Centre for Magnetic Resonance, University of Queensland, Brisbane, Queensland 4072, Australia.
5 Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204.
6 ZMBH-Center for Molecular Biology, Heidelberg, University of Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany.
7 Genetics and Aging Unit and Department of Neurology, Harvard Medical School, Massachusetts General Hospital, Charlestown, Massachusetts 02129.
9 Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92717.
10 Department of Pathology, University of Melbourne, and Neuropathology Laboratory, Mental Health Research Institute of Victoria, Parkville, Victoria 3052, Australia.
Methods: hydrogen peroxide assay. The colorimetric H2O2 assay was performed in a 96-well microplate (SpectraMax Plus, Molecular Devices Corp.), according to a modification of an existing protocol. Polypeptides (10 µM) or vitamin C (10 µM), Cu(II) (1 µM), and a H2O2 scavenging agent, tris(2-carboxyethyl)phosphine hydrochloride (Pierce, 50 µM), were co-incubated in PBS buffer (300 µL), pH 7.4, for 1 hour at 37 °C. Following incubation, the unreacted tris(2-carboxyethyl)-phosphine hydrochloride was detected by 5,5´-dithiobis(2-nitrobenzoic acid) (Sigma, 50 µM). The amount of H2O2 produced was quantified based on the formula: H2O2 (µM) = A x 106/(2 x L x M), where A is the absolute absorbance difference between a sample and catalase-only (Sigma, 100 units/mL) control at 412 nm; L = the vertical pathlength, corrected automatically by the plate reader to 1 cm; M is the molecular absorbance for 2-nitro-5-thiobenzoate (14,150 M-1 cm-1 at 412 nm).
inorganic phosphate
Two active forms of UDP-N-acetylglucosamine enolpyruvyl transferase in gram-positive bacteria
J. Bacteriol. 182: 4146–52 (2000).
Wensheng Du, James R. Brown, Daniel R. Sylvester, Jianzhong Huang, Alison F. Chalker, Chi Y. So, David J. Holmes, David J. Payne, and Nicola G. Wallis.
Anti-Infectives Research, SmithKline Beecham Pharmaceuticals, Collegeville, Pennsylvania 19426.
Methods: detection of inorganic phosphate. The activity of MurA1 and MurA2 was assayed by measuring the release of Pi from the UDPAG and PEP reaction, using a malachite green assay in 50 mM HEPES (pH 7.5) at room temperature. A typical assay of 200 µL contained substrates and enzyme (MurA1 at 200 nM or MurA2 at 50 nM). For kinetic measurements, the assay was performed with a five-by-five array of various substrates on a half-area 96-well microtiter plate (Costar 3696; Corning Inc., Corning, N.Y.) with a SpectraMax Plus plate reader (Molecular Devices Corp.)
Microdetermination of phosphorus using the SpectraMax Plus microplate spectrophotometer: choice of microplate, cuvette or test tube assay formats
Molecular Devices MaxLine Application Note #24.
This application note details two methods for measuring inorganic phosphorus using Molecular Devices’ SpectraMax Plus microplate spectrophotometer. The first method uses a commercial kit obtained from Sigma Diagnostics, based on the original Fiske-Subbarow chemistry, and offers the convenience of ready-made reagents. The second method is approximately 10 times more sensitive, and thus is useful for samples containing 0.1 to 5.0 µg Pi/mL. Whichever method is used, the SpectraMax Plus microplate spectrophotometer offers the versatility of making the absorbance measurements in microplates, in standard 1 cm cuvettes, or in 12 x 75 mm test tubes.
reactive oxygen species
Superoxide anion production from human neutrophils measured with an improved kinetic and endpoint microassay
J. Immunol. Methods 142: 95–104 (1991).
E. Sabrinah Chapman-Kirkland, James S. Wasvary, and Bruce E. Seligmann.
Research Department, Ciba-Geigy Corporation, Summit, NJ 07901 USA.
Summary. Superoxide dismutase (SOD)- inhibitable reduction of cytochrome c is widely used to measure superoxide production by neutrophil cells and cell fragments. The authors report using a dual wavelength measurement and novel modifications to the microplate assay to lessen the perturbations caused by the microplate and improve experimental reproducibility. A new surface modified microplate (Plastek A*) was compared to untreated microplates. The modified surface prevented the adherence and consequent activation of PMNs. Neutrophils in the two types of plastic microplates were shown to have a statistically significant difference in basal as well as stimulated levels of superoxide production. Absorption measurements were made using dual wavelength measurement at 550 nm (1 nm bandwidth), the absorption maximum of reduced cytochrome c, and 557 nm (1 nm bandwidth), an isosbestic point. The authors observed that the difference values (550–557 nm) significantly increased reproducibility and sensitivity (detection limit) as compared to the single wavelength measurement at 550 nm.
Kinetic microplate assay for superoxide production by neutrophils and other phagocytic cells
Methods in Enzymology 196: 567–575 (1990).
Laura A. Mayo and John T. Curnutte.
Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, CA 92037 USA.
Summary. This review describes methods of isolation and use of human neutrophils in assays for superoxide production. The kinetic microplate assay for superoxide follows the reduction of cytochrome c at 37 °C in a Molecular Devices microplate reader fitted with a special narrow (1 nm) bandwidth 550 nm interference filter. Single well illumination and use of AUTOmix in whole cell assay are reported to be valuable. Analysis of nonlinear kinetic data with SoftMax Pro software is presented.
chemical measurements: luminescence
ATP
ATP quantitation in the LMax™ microplate luminometer
Molecular Devices MaxLine Application Note #40 (2000).
Evelyn McGown, Ph.D. and Michael Su, M.S.
Summary. The LMax microplate luminometer gives a detection limit of about 0.02 fmol ATP/well and a dynamic range of 5 logs with the ENLITEN® ATP assay system from Pomega Corp. These results are as good as, if not better than, results obtained using a standard tube luminometer. The LMax offers the advantage of a 96-well format and automated reagent addition for better precision and higher throughput. In addition, SoftMax Pro for LMax provides a powerful and convenient instrument control and data calculation package.
reactive oxygen species
Measuring oxidative bursts with the photoprotein pholasin
Communications in Clinical Cytometry 46: 324 (2001).
Doug Redelman1, Qiao Zhong2, Dorothy Hudig2, Linda M. Castell3, Don Roberts4 and Wayne Y. Ensign4.
1 Sierra Cytometry/UNR Cytometry Center, Reno NV.
2 University of Nevada, Reno NV.
3 University of Oxford, UK.
4 Naval Health Research Center, San Diego, CA.
Methods: kinetic assay for superoxide anion. The oxidative burst stimulated by formyl-methionyl-leucyl-phenylalanine (fMLP) was measured in a kinetic chemiluminescence assay (ABEL, Knight Scientific Ltd., Plymouth, UK). The chemiluminescent agent in this system is the photoprotein from the mollusk Pholas dactylus (Pd-prot or Pholasin). The 96-well microplate assay kit includes blood dilution medium, Pd-prot, fMLP, phorbal ester (PMA), and Adjuvant-K, a proprietary luminescence-enhancing agent. To perform the assay, whole blood was diluted 1:100 (20 µL + 20 mL) in the provided diluent. The wells of the white opaque microplates had diluent, Pd-prot and the enhancing agent plus 200 µL of 1:100 diluted whole blood in a total volume of 175 µL. The reactions were set up as sets of 8 wells (4 duplicate blood samples) at a time in order to minimize the effects of prolonged standing. The plates were read in a kinetic luminometer (LMax, Molecular Devices Corp.) Two types of readings were used. First, the wells were measured in “slow kinetics” mode in which the reading head moved from well to well to determine the background from each well over a 5-minute period. The stimulated responses were read in “fast kinetics” mode in which the reading head remained positioned over a single well for the duration of the assay (100 seconds). The response was initiated by injecting 25 µL fMLP solution to produce a final concentration of ~1 µM fMLP in the 200 µL total reaction volume. The response was measured once per second and the data are reported as “relative light units” (RLU). SoftMax Pro software was used to process the data.
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