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General Methods
Enzyme Linked Immunosorbent Assay
Protein Quantitation
Nucleic Acid Quantitation
Gene Expression
Instrumentation Analysis
enzyme linked immunosorbent assay
absorbance
A microtiter-based assay for the detection of protein tyrosine kinase activity
Anal. Biochem. 190: 249–253 (1990).
Jeffrey S. Cleaveland, Peter A. Kiener, David J. Hammond and Bernice Z. Schacter.
Bristol-Myers Squibb, Wallingford, CT 06492 USA.
Summary. A rapid, nonradioactive alternative assay has been developed to screen for protein tyrosine kinase (PTK) inhibitors. This assay uses a substrate polymer adsorbed to a microplate which can be phosphorylated by PTK. The amount of phosphotyrosine produced is determined in an enzyme-linked immunosorbent assay (ELISA) using an antibody to phosphotyrosine. Color development is shown to be dependent upon assay time, enzyme, ATP, and substrate concentrations. Specific PTK inhibitors reduced the total phospho-tyrosine incorporation into the substrate polymer. Results obtained in the ELISA compare with those obtained by direct phosphorylation of the substrate with [32P]ATP.
Pathogenesis of herpes simplex virus-induced ocular immunoinflammatory lesions in B-cell-deficient mice
J. Virol. 74: 3517–24 (2000).
Shilpa P. Deshpande, Mei Zheng, Massoud Daheshia, and Barry T. Rouse.
Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37996-0845.
Methods: HSV ELISA. To assess the role of virus replication in lesion development, mice were infected on the cornea and 5 days later were given intravenously 300 µL of anti-HSV serum (36.5 µg/mL; HSV-specific IgG). Sera were collected from HSV-1 re-immunized BALB/c mice and checked for HSV-specific total IgG by standard enzyme-linked immunosorbent assay (ELISA) as described previously. Briefly, the ELISA plates were coated with 100 µL of HSV antigens or anti-mouse IgG (1 µg/mL; PharMingen) as a standard in carbonate buffer (pH 9.8) overnight at 4 °C. Serum samples were diluted 1:200 in phosphate-buffered saline (PBS) and run in triplicate with purified mouse IgG as a standard (PharMingen), followed by horseradish peroxidase-conjugated goat anti-mouse IgG (PharMingen). Quantification was performed with a SpectraMax microplate reader (Molecular Devices Corp.)
luminescence
High sensitivity, wide dynamic range, horseradish peroxidase (HRP) ELISA using the LMax™ microplate luminometer
Molecular Devices MaxLine Application Note #41 (2000).
Evelyn McGown, Ph.D. and Michael Su, M.S.
Summary. Molecular Devices LMax microplate luminometer provides an easy and sensitive means of measuring chemiluminescent ELISAs in a microplate format. This particular work was done with a commercial kit for human TNF-a; however, excellent results would also be expected with other optimized HRP-based ELISA assays using luminol/peroxide substrate.
Methods: HRP assay. Materials included the Quantikine Human TNF-a Immunoassay kit (R&D Systems, Cat #DTA50). The kit contains all reagents and standards needed for the assay, including the polystyrene microplate (12 strips of 8 wells/strip) pre-treated with antibody against TNF-a.The kit instructions were followed for preparation of the reagents and substrate solution. The TNF-a stock standard was reconstituted with 0.5 mL deionized water, gently vortexed and allowed to stand for 15 minutes at ambient temperature. The working standards were prepared by making a serial 1:10 dilution of the stock standard in calibrator diluent (QDP5). The concentrations of the working standards ranged from 7000 pg/mL to 0.07 pg/mL.
After first putting 50 µL assay diluent (QD1-27) into wells of the pre-treated microplate (supplied with the kit), working standards of TNF-a and the blank (QDP5) were pipetted in triplicate (200 µL each). The kit instructions were followed for the subsequent aspiration, washing, incubation with conjugate, aspiration and washing steps. After the final step, the addition of substrate solution, the wells were covered with an adhesive strip and incubated with shaking at ambient temperature for 30 minutes. The adhesive cover was removed and the microplate was placed into the LMax. To ascertain the optimal integration time, the plate was read at 0.1, 1, 5 and 10 seconds per well.
protein quantitation
absorbance
PathCheck® applied to measurement of protein solutions in the SpectraMax® Plus microplate spectrophotometer
Molecular Devices MaxLine Application Note #26.
Protein concentration is commonly estimated by measuring the absorbance at 280 nm (A280) and calculating the concentration using the extinction coefficient of the protein. The procedure is feasible because extinction coefficients are typically obtained using standard cuvettes with a fixed 1 cm pathlength. Extinction-based protein assays can also be done in microplates, but they have not been widely used because of the inconvenience of compensating for variable pathlengths of samples in microplate wells. The introduction of the Molecular Devices’ SpectraMax Plus microplate spectrophotometer marks the first time that photometric measurements made in a microplate format can be automatically normalized such that the resultant values are identical to those obtained using the corresponding solutions in a standard 1 cm pathlength cuvette. This application note outlines the use of the pathlength correction feature “PathCheck”, compares absorbance results obtained in cuvettes and microplate, and gives an example in which PathCheck is applied to column chromatographic fractions and the elution profile is displayed.
fluorescence
Using the NanoOrange Protein Quantitation Kit in the ƒMax microplate fluorometer
Molecular Devices MaxLine Application Note #22.
Summary. This application note describes how to use the NanoOrange® Protein Quantitation Kit from Molecular Probes in the ƒMax fluorescence microplate reader with SoftMax® Pro software from Molecular Devices Corporation. This assay is much more sensitive than traditional photometric methods such as A280, BCA, Bradford, or Lowry assays in the microplate format. The dynamic range of this assay in microplate format is 100 ng/mL to 10 µg/mL. The assay is read with an excitation wavelength of 485 nm and an emission wavelength of 590 nm. Both the excitation and emission filters are standard on the ƒMax fluorescence microplate reader. The kit from Molecular Probes contains sufficient reagent for 2000 assays using a 200 µL volume. The data presented in this application note confirm the dynamic range and lower limit of detection described by Molecular Probes in their Application Note (MP 6666). The data presented here are preliminary and do not represent a fully optimized assay.
nucleic acid quantitation
ultraviolet
DNA and RNA measurements in SpectraMax microplate spectrophotometers
Molecular Devices MaxLine Application Note #33 (1999).
Evelyn McGown, Ph.D.
Summary. This application note provides guidelines for optimizing DNA/RNA absorbance measurements in SpectraMax microplate spectrophotometers. Topics include recommendations for best quality DNA absorbance values, using Molecular Devices microplate spectrophotometers’ PathCheck feature to normalize absorbance values to a 1-cm pathlength, options for eliminating microplate OD in PathCheck calculations, DNA/RNA estimation using absortivity, and using SoftMax® Pro to calculate DNA concentration.
UV absorbance measurements of DNA in microplates
Biotechniques 28: 60-64 (2000).
Evelyn L. McGown.
Molecular Devices Corporation, Sunnyvale, CA USA
Summary. With attention to technique, accurate and reproducible DNA measurements can be made easily in microplates. The lower limits of detection and quantitation are comparable to those obtained with semi-micro cuvettes in conventional spectrophotometers.
fluorescence
Using the OliGreen® Oligonucleotide Quantitation Reagent in an ƒMax microplate fluorometer
Molecular Devices MaxLine Application Note #21.
Summary. This application note describes how to use OliGreen Oligonucleotide Quantitation Reagent from Molecular Probes in an ƒMax fluorescence microplate reader with SoftMax® Pro software (both from Molecular Devices Corporation). Single-stranded DNA and oligonucleotides are traditionally measured at 260 nm. OliGreen has two advantages over the traditional method: greater sensitivity and specificity. Molecular Probes describes a dynamic range of 1 ng/mL to 1 µg/mL and sensitivity of 200 pg/200 µL in their application note on the use of OliGreen (MP 7582 02/08/96). This assay is an endpoint assay read using an excitation wavelength of 485 nm and emission wavelength of 538 nm. A single concentration of the OliGreen dye is used for the entire dynamic range. The OliGreen kit provides enough reagent in a single kit to perform approximately 2000 samples in a volume of 200 µL each.
luminescence
DNA quantitation in the LMax™ microplate luminometer
Molecular Devices MaxLine Application Note #37 (2000).
Evelyn McGown, Ph.D. and Michael Su, M.S.
Summary. The LMax microplate luminometer detects a lower limit of 5 pg DNA/well with the DNAQuant™ DNA quantitation system (Promega Corporation, Product No. K4000). These results are as good as, if not better than, the results obtained using a standard tube luminometer.The LMax microplate luminometer also offers the additional advantage that adding luciferin/luciferase reagent and the subsequent read can be automated so that variability due to differences in timing of the flash luminescence reaction can be eliminated.
SNPs: fluorescence
Measurement of molecular beacons in the Gemini spectrofluorometer
Molecular Devices MaxLine Application Note #36 (1999).
Evelyn McGown, Ph.D. and Michael Su, M.S.
Summary. This application note describes measurement of probe/target complexes with the Gemini microplate spectrofluorometer. Samples of molecular beacons and complementary targets were obtained from Research Genetics, Huntsville, Alabama. The microplates used were Nunc brand, black 384-well microplates, surface-treated to minimize binding (cell culture-treated). All samples were prepared in 10 mM Tris buffer, pH 8, containing 1 mM MgCl2, and the final concentrations of beacon and target were 0.3 mM and 1.6 mM, respectively, in a total volume of 54 µL. The reaction mixtures were incubated at least 30 minutes at ambient temperature before measurement in a Gemini microplate spectrofluorometer. With its dual scanning monochromators, it is easy to optimize excitation and emission wavelengths for specific fluorophores and to customize the settings for combinations of probes. Mixtures of 2, 3, and (probably) 4 probes in a single mixture can be resolved.
gene expression
luminescence
Dual-luciferase quantitation in the LMax™ microplate luminometer
Molecular Devices MaxLine Application Note #39 (2000).
Evelyn McGown, Ph.D. and Michael Su, M.S.
Summary. Colorimetric reporter assays have been widely used, but bioluminescent reporter systems, especially using firefly luciferase, are becoming increasingly popular because of their speed, sensitivity and wide dynamic range. Promega Corporation has recently introduced their Dual-Luciferase® Reporter Assay System which utilizes the firefly (Photinus pyralis) and Renilla (Renilla reniformis) luciferases. Because the enzymes have different substrate requirements, they can both be measured in a single reaction mixture. The firefly luciferase is typically used as the experimental reporter, and the renilla luciferase serves as the control. Firefly luciferase enzyme catalyzes the oxidation of luciferin with the concomitant release of light. The reaction requires ATP, Mg+2 and O2. Renilla luciferase catalyzes the O2-dependent oxidation of coelenterate luciferin (coelenterazine) but does not require Mg or ATP. Both reactions can easily be measured in Molecular Devices’ LMax microplate luminometer. An example of results is presented in this application note.
instrumentation analysis
absorbance
Using the SpectraMax® Plus for USP dissolution calibration
Molecular Devices MaxLine Application Note #27.
Summary. Calibration of a dissolution apparatus is necessary to produce accurate results for dissolution testing of pharmaceutical dosage forms and to allow comparison between batches and between laboratories. USP requirements for calibration are given in the Apparatus Suitability Test in USP <711> Dissolution. USP <711> specifies that both prednisone and salicylic acid are to be measured by UV spectrophotometry (242 nm and 296 nm) by comparison to a standard solution of known concentration. (Compendial requirements for UV spectrophotometry are given in USP <851> Spectrophotometry and Light Scattering). This application note describes how spectrophotometric analyses for dissolution calibration can be quickly and conveniently carried out in a 96-well format using the SpectraMax Plus microplate spectrophotometer. The PathCheck feature of the SpectraMax Plus automatically corrects the results to a 1 cm pathlength; therefore, samples read in a microplate give the same values that they would if read in a standard cuvette. Accurate pipetting of the samples into the wells is not necessary; indeed, PathCheck corrects for pipetting errors. Using flexible custom formulas and the spreadsheet capability of SoftMax Pro® software, the data are calculated automatically, and final results are displayed as overall PASS/FAIL for each apparatus.
Verifying multichannel pipettor performance with standard dispense solutions in the SpectraMax® Plus
Molecular Devices MaxLine Application Note #28.
Summary. Two common methods for checking precision and accuracy of pipettors are gravimetric and spectrophotometric. Ideally, pipettor performance should be checked using the actual dispense reagent as the calibrator solution. However, most dispense reagents are colorless, and an absorbance measurement cannot be obtained in the visible range (400–750 nm) without the addition of a dye. The SpectraMax Plus microplate spectrophotometer uses the near infra-red absorbance of water to determine the pathlength in each well, which is proportional to volume. The dispensed volumes are calculated by reference to a pathlength/volume standard curve (prepared separately with a certified pipettor). The standard curve can be prepared once for a given dispense reagent and batch of microplates and stored for subsequent use. This application note describes how to check the performance of multichannel pipettors with the SpectraMax Plus. Pathlengths in the wells are measured directly and volumes are calculated automatically. The performance can be checked using the same buffers or reagents that are being dispensed by the pipettor, eliminating possible discrepancies in pipettor performance between normal dispense reagent and calibrator solution. Because initial separate calibration of one channel is not necessary, the method is suitable for checking performance of automated pipetting stations, including 96-channel pipettors.
UV absorbance measurements in SpectraMax microplate spectrophotometers
Molecular Devices MaxLine Application Note #32 (1999).
Evelyn McGown, Ph.D.
Summary. Despite the huge popularity of microplates, many people experience difficulty when adapting assays to them. One source of confusion is the fact that samples read in a cuvette and in a microplate do not have identical raw absorbance values because of differing optical pathlengths. Also, microplates are susceptible to surface effects including floating particulates, foaming and variable meniscus formation. The susceptibility to particulates has increased in recent years because modern microplate spectrophotometers such as SpectraMax are designed to accommodate microplates with smaller wells and thus have smaller beams than older instruments. All of these factors cause microplates to demand more attention to careful technique to get accurate absorbance results. Especially in the UV spectral range, appropriate procedures should be followed to avoid poor reproducibility and unsatisfactory sensitivity. This application note provides guidelines for optimizing UV measurements in SpectraMax microplate spectrophotometers, particularly measurements of proteins at 280 nm and nucleic acids at 260 nm.
fluorescence
Selecting excitation and emission wavelengths using the Gemini microplate spectrofluorometer — basic principles
Molecular Devices MaxLine Application Note #30.
Summary. This application note gives a basic procedure for optimization of excitation and emission wavelengths of the Gemini microplate spectrophotometer. An example is given for application of the procedure to a fluorophore with a relatively large Stokes’ shift (quinine). The first step in developing fluorescence analysis methodology is to select the excitation wavelength. The next step is to select the optimum combination of emission wavelength and cutoff filter that gives the highest possible signal/background ratio. Generally, samples containing 10-8 M to 10-6 M fluorophore will give sufficient signal. For optimal signal/background results, data should be acquired with the highest PMT (voltage) setting. Thus, the fluorophore concentration should be low enough that the emission scans can be done with high PMT without saturating the detector.
Optimizing excitation and emission wavelengths for narrow Stokes’ shift fluorophores using the Gemini and SoftMax® Pro
Molecular Devices MaxLine Application Note #31.
Summary. When optimizing excitation and emission wavelengths, the easiest case is when the fluorophore has a relatively large Stokes’ shift (> 80 nm); the optimal wavelengths are those giving maximal signal, assuming no background interference. If the Stokes’ shift is narrow, the selection/optimization process is less straightforward because scattered excitation light interferes with the fluorescent signal. This application note gives details of an optimization procedure for fluorophores with Stokes’ shifts less than 80 nm and includes custom SoftMax Pro formulas to assist in the selection process. The fluorophore used in this example is fluorescein.
Sensitivity test for Molecular Devices microplate fluorometers
Molecular Devices MaxLine Application Note #45 (2001).
Evelyn McGown, Ph.D. and Michael Su, M.S.
Summary. This application note contains the recommended procedure for verifying the sensitivity of the Gemini, Gemini XS, the FlexStation™ and the ƒMax® in fluorescence mode (top read). If they are functioning normally, and there is no operator error, this test will demonstrate the sensitivity of the instrument in 96-well format. For the purposes of this application note, sensitivity is defined as the amount of fluorescein (in femtomoles/well) giving a signal equal to or greater than 3 standard deviations of the signal from the background wells (i.e., the wells filled with buffer.) The test is done with 0.2 mL/well, so the fmol/well expression can be translated to concentration (picomolar) by multiplying by 5. The calculations are made from fluorescence measurements of 2 microplates, one filled with 100 nM fluorescein and the other filled with buffer.
AquaMax™ 1536 dispenser accuracy and precision testing
Molecular Devices AquaMax Application Note #1 (2000).
Gayle Teixeira.
Summary. This application note is intended to provide a basic procedure for testing accuracy and precision. The AquaMax 1536 dispenser has been designed to achieve a 1.0 µL volume dispense with an accuracy of ± 5.0% and precision of less than 10% CV.
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