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We dried 100 μg of protein in a centrifugal evaporator and prepared the sample for mass spectrometry according to the iTRAQ reagent protocol (SCIEX, Framingham, MA).
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Fifty known-age, adult seals (9 27 years, 24 males, 26 females) in McMurdo Sound were sampled for mass, total body fat, blubber depth and a suite of blood parameters (21 variables) to assess hydration state, nutritional plane, reproductive hormones (females only), organ function and immune status.
Samples for mass spectrometry were prepared as previously described [30].
In order to prepare the samples for mass spectroscopy analyses, the active fraction of mini-S column was subjected to SDS-PAGE using 12.5% acrylamide gels.
The materials and methods used to prepare the protein samples for mass spectrometry were fully described for the identical set of samples elsewhere [39]: briefly, each sample was lysed, extracted into global, soluble, and insoluble fractions then trypsinized; and further fractionated by ion exchange chromatography.
SKS and VNP processed the samples for mass spectrometry and VNP performed mass spectrometry.
Samples for mass spectrometry were desalted by ethanol precipitation from ammonium acetate.
Preparation of samples for mass spectrometry were performed by the Mass Spectrometry Core Facility at University of Texas Medical Branch, Galveston.
A key component of these studies is the method used to prepare selectively targeted protein samples for mass spectrometry interpretation.
The gel digestion of selected samples for mass spectrometry was done according to Hanna et al. [ 25] with some modifications.
Samples for mass isotopomer analysis of intracellular metabolites were taken, processed, and analyzed as described by Zhao et al. (2008).
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