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A total of 10,000 events were accumulated for each analysis; samples were analyzed in triplicate.
To reduce any bias introduced prior to analysis, samples were analyzed in random order.
For comprehensive neurochemical analysis, samples were analyzed as previously described but with a 6 min HPLC gradient (Song et al., 2012).
For C. parvum analysis, samples were analyzed using EPA method 1623 for Cryptosporidium and Giardia in water by filtration, immunomagnetic separation, and fluorescent-labeled antibodies.
Prior to microarray hybridization analysis, samples were analyzed for enrichment relative to input DNA, which is total DNA extracted before immunoprecipitation, by quantitative PCR (qPCR) with primers specific for the Ss-lrpB control region (Additional file 1: Dataset S1).
HPLC analysis: Samples were analyzed by a LC 2010 analytical HPLC system (Shimadzu, Kyoto, Japan) equipped with a Varian Inertsil RP C18 column (ODS-3, 5 μm, 150×4.6 mm; Agilent) and eluted with various mixtures of acetonitrile/water containing trifluoroacetic acid (0.1 %) under isocratic and gradient conditions (flow rate 1.0 mL min−1, detection 254 nm).
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To determine the day-to-day repeatability of the analysis, the samples were analyzed by capillary electrophoresis for three consecutive days, performed as sequence of five runs each day.
For compositional analysis, the samples were analyzed as outlined in Ibáñez and Bauer [ 28].
(19) For intact protein analysis, myoglobin samples were analyzed by high resolution Fourier-transform (FT) mass spectrometry (MS) on a Thermo LTQ-Orbitrap XL mass spectrometer 33) (ThermoFisher).
LC-MS/MS Analysis The samples were analyzed via electrospray tandem mass spectrometry (LC-MS/MS) on a Thermo LTQ Orbitrap XL, using a 60,000 RP survey scan, m/z 375-1950, with lockmasses, followed by 5 LTQ CAD scans on doubly and triply charged-only precursors between 375 Da and 1500 Da.
Therefore, our study was sufficiently powered to detect a 1.4-fold change difference in the combined analysis and a 2-fold change in the subgroup analysis where FF and FFPE samples were analyzed separately.
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