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After centrifugation, 159 g of YH was separated into two phases, 132.3 g of supernatant (YHL, 83.8%) and 24.6 g of slurry (YHS, 16.2%).
A portion (2 g) of supernatant was mixed with 0.5 g of 1.2 mol/L HCL, heated at 95°C for 65 min without charring, and left to cool at room temperature for 15 min.
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After centrifugation of homogenates at 15,000 g, 10 μg of supernatant proteins were used to determine Nqo1 activity as previously described using DCPIP as a substrate [ 11].
After two rounds of washing (centrifugation for 20 min at 1500 g, replacement of supernatant with sterile Volvic), a final volume of 1.5 ml was vortexed for 30 sec in a 2 ml plastic tube, together with several 2-mm glass beads, to grind up the dead paramecia.
The slowly-sedimenting membranes generated were separated from the donor membrane by a medium speed centrifugation (20K × g ) and collected by high-speed sedimentation (100K × g ) of the supernatant fraction (20KS).
After 4 h of sample rotation at 4 °C and centrifugation for 5 min at 1000 g, 200 μl of supernatant were again mixed with 100 μl of protein A/G PLUS-agarose and subjected to rotation over night.
speed in a tabletop centrifuge and the supernatant was precipitated with solid ammonium sulfate (0.3 g per ml of supernatant).
After centrifugation (72 g), 50 µl of supernatant were transferred to 96-well plates and the amount of hemoglobin released by virus-cell fusion induced hemolysis was determined by the measurement of optical density at 405 nm.
After centrifugation at 16,000× g 20 μl of supernatant was applied on the gel for analysis.
After incubation for up to 60 min at room temperature the tubes were centrifuged (5 min, 490 g), 450 μl of supernatant was removed and both supernatant and pellet were counted in a Wallac 1282 Compugamma Universal Gamma Counter.
After 1-h ultracentrifugation at 150,000× g, the aliquots of supernatant and pellet fractions were analyzed by SDS-PAGE and Western blot to assess the amount of actin (β- and γ-isoforms) and β-tubulin.
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