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To quantify eicosanoids in the cell lysate, lipid standards and internal standards mixture were spiked into 0.2 mL of Tris-HCl buffer.
Aliquots of the digest mixture were spiked into each food matrix and analysed to evaluate the effects of the food matrix on the MS detection.
Aliquots of the SIS peptide mixture were spiked into the lyophilized peptide samples, followed by reconstitution in 350 μl of 0.01 M (NH4 HCO3, incubation on an orbital shaker for 15 min at RT, reduction using 30 μl of 0.05 M TCEP, incubation for 1 hour at RT, and dilution in 375 μl of 90%water/10%acetonitrile/0.2%2% trifluoroacetic acid.
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After the incubation, each mixture was spiked into a serum aliquot.
In the second experiment, bovine β-casein was mixed with each food matrix first, then each sample mixture was spiked with an IS (IS1 or IS2) separately.
In a competition assay, the mixture was spiked with a range of concentrations of a free inhibitor interfering with the capture of HDAC complexes by the immobilized inhibitor.
Mixtures were spiked in triplicate in the range of LOQ and 10 LOQs.
The identities of the metabolites were compared with known standards, and the reaction mixtures were spiked with the appropriate standard metabolites.
Following trypsinization, equal quantities of tryptic peptide mixtures were spiked with 1 pmol of synthetic (Glu1 -Fibrinopeptide B (Glu1 -Fibrinopeptidech) to serve in the downstream analysis as an internal control for the efficiency of individual laBelinGluFibtions.
The multiple libraries of BLM-1 and BLM-2 mixtures were spiked with either of these pools at one of three (high, low, medium) concentrations.
Briefly, unfertilized soil was mixed with compost at a ratio of 3 1 (fw/fw) and replicates of soil-compost mixture and fertilized soil were spiked with [13C6]-pyrene to a final concentration of about 100 mg kg−1 following the procedure described previously (for details see Adam et al. 2015; Kästner and Mahro 1996).
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