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After 10 min, the sample was dried using a vacuum dryer.
After mild ultra-sonication for about 30 60 min, the sample turned transparent.
After 15 min the sample was passed through a filter paper.
After centrifugation at 4000g for 15 min, the sample was washed repeatedly with water, and dialyzed against deionized water using a 1 kDa cutoff membrane.
The solution was first kept in the dark (from −240 min); at −180 min, the sample was immersed and kept in the dark (up to 0 min).
After centrifugation (5,000 × g) for 15 min, the sample was repeatedly washed with water until a negative test against AgNO3 was obtained.
Similar(19)
For imaging of the reconstituted hemichannels, proteoliposomes were deposited on freshly cleaved mica (20 μL) and allowed to attach for 30 min. The sample was heated for 5 min at 37 °C to induce liposomal fusion.
17 The subjects were monitored for 20 min while sitting at rest for 5 min. The sample frequency was 1000 Hz per channel.
The nuclear suspension was stirred vigorously on ice for 30 min. The sample was centrifuged at 15 000 rpm for 12 min at 4°C, and the nuclear/membrane extract was frozen and stored at −80°C.
The resulting membrane enriched fraction was re-suspended in 500 µl lysis buffer plus 1% Triton X-100 and incubated on ice for 15 min. The sample was re-centrifuged at 13000 rpm for 10 min, and subjected to SDS-PAGE.
A 4% Na deoxycholate solution was made fresh (125 mM NaCl, 4% Na deoxycholate) and added to a final concentration of 0.05% followed by an incubation at room temperature for 10 15 min. The sample was sonicated for 5 one minute cycles with a 0.5 sec pulse at 60% output and then centrifuged at 16,000 rpm (38400 x g) for 110 minutes.
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