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Extracts were centrifuged and the supernatant (nuclear extracts) transferred to pre-chilled tubes for storage at -70°C.
Approximately 25 µl of ice-cold nuclear extraction buffer was added to the pellets and incubated for 30 min. Extracts were centrifuged and the supernatant (nuclear extracts) transferred to pre-chilled tubes for storage at -70°C.
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The hexane/acetone extraction was repeated twice and the extract transferred to the appropriate volumetric flask.
Single crystals were extracted, transferred to the reservoir condition supplemented with either 20% glycerol (4JN8) or 20% ethylene glycol (4JN7), and vitrified by being plunged in liquid nitrogen.
Homogenate was centrifuged (2000 × g for 10 min) and the supernatant (HE extract) transferred to vials and stored (-20°C) until assayed.
Extraction of the lipophilic phase (LE) was made on pellet by adding acetone (1 4 w/v), and centrifuging at 2000 × g for 15 min. The supernatant (LE extract) transferred to vials and stored (-20°C) until assayed.
Extracts were centrifuged, and the supernatant (nuclear extracts) was transferred to the prechilled tubes for storage at −80°C.
The extracts were mixed thoroughly and fifty micro liters of extracts were transferred to well 2 to well 8 from which fifty micro liters were discarded.
Briefly, total protein extracts were prepared from kidney medulla samples, separated on SDS polyacrylamide gel (30 μg of protein extracts) and transferred to HybondTM nitrocellulose membrane (Amersham).
The obtained extracts were transferred to 50 mL volumetric flasks, filled up to their volume with extractant, and subjected to the HPLC analysis.
Dissolved extracts were transferred to a 5-mm NMR tube for analysis.
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