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50µg of each protein extract were transferred to a new tube and used later for iTRAQ analysis and the remained extract was prepared for the 2-D PAGE analysis.
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Fungal assays: Malt agar gels (4.8 % w/v) containing the remaining extract (i.e. from 4.8 g of dry wood) were prepared so that the amount of extract in each gel corresponded to the extractive content of the wood sample.
In addition, the analytes were re-concentrated prior to inject into the gas chromatography (GC) inlet because of the organic solvent removing from the remaining extract of MSPE technique.
The remaining extract was finally dried by desiccator.
A 200 μl aliquot was taken for purification and the remaining extract was stored at −80°C.
The remaining extract was used to determine protein concentration (Bradford, 1976), with BSA as the standard.
The remaining extract was concentrated to approximately 1 2 mL in a Kuderna-Danish apparatus followed by nitrogen evaporation.
Subsequently, the remaining extract was stored and if analytical results exceeded the top calibrator, second fecal extracts were further diluted 1 6 with incubation buffer and assayed again.
Reducing sugars in the extract, which could potentially interfere with subsequent enzyme activity assays, were removed by passing the remaining extract through a PD10 desalting column.
To remove acetonitrile the plate was dried overnight in a fume cupboard and the remaining extract was dissolved in only 2 μL DMSO the next day.
A subsample (5%) was retained for gravimetric lipid determination and the remaining extract was concentrated to allow for transfer of the extract and rinsing of the flask into a 10 ml volumetric flask.
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