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The most important mono- and di-saccharides were analysed by flow injection analysis and a mixture was submitted to high performance anion chromatography with amperometric detection, using the developed device as the working electrode.
The resulting mixture was submitted to an ultrasonic bath for 10 min, where a solution of sodium borohydride was added under stirring in one portion at room temperature.
The digestion was terminated with formic acid, and 2 μg of the islet tryptic peptides mixture was submitted to LC-MS to verify the digestion efficiency.
Finally, the protein mixture was submitted to chromatography through a Sephacryl S200 column and factions containing only the 20 kDa protein were pooled and analysed by SDS-PAGE and Western blot.
The mixture was submitted to 35 cycles and to a final extension step of 7 min, at 66°C for cytb and COI and at 70°C for ND5.
The mixture was submitted to a thermostatic bath (temperature variable according to the factorial design) for 10 min for medium adaptation, after 5 g of fermented bran containing the cellulolytic complex or 5 mL of commercial enzyme with dilution of 1 : 100 (v/v) was added.
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After that, 20 μg of the mixture were submitted to cDNA synthesis with the Superscript III kit (Invitrogen, Carlsbad, CA, USA), following the manufacturer's instructions.
Chemically activated anthracites with pore textures optimised for methane storage were densified by mixing suitable granular fractions, and the prepared mixtures were submitted to increasingly high mechanical pressures (up to 220 kg cm−2).
The peptide mixtures were submitted to MALDI-TOF MSfor identification.
These mixtures were submitted to western blotting and detected by NB509-11G8 andibody and X53.
The gel mixtures were submitted to a DIN/ISO 10993-4, -5, 10, -11 biocompatibility testing (including gels prepared from human albumin and of the test species mouse, rabbit, rat, and guinea pig) by BIOSERV Analytik & Medizinprodukte GmbH (Rostock, Germany) and passed successfully.
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