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Assuming that this protein is due to lysis of 40 mg of cells and that the specific cell volume is 2 ml/g cells, the intracellular concentration of, for example, 2-oxoglutarate should have been as high as 750 mM". Specific cell volume, which is the ratio of cell volume per cell dry weight, is not constant but depends on growth rate and physiology.
The amount of glutathione was expressed as nmol per mg of cells.
Total protein content was extracted from 5 mg of cells as described previously (De Marsac and Houmard [1988]).
A serial transfer of 50 mg of cells (dry weight) into fresh medium was performed every 24 h.
Therefore, 30 50 mg of cells harvested in the late exponential growth phase were resuspended in 2 mL of minimal media.
The cells were collected and weighed, and 250 mg of cells were then suspended in 100 μL of a TES buffer (50 mM tris HCl, pH 8, 1 mM EDTA, 25% sucrose).
Similar(29)
For immunoprecipitation of over-expressed proteins in fibroblasts, 4 mg of cell lysate was incubated o/n with protein G beads coated with anti-FLAG antibody or GFP Trap_MA beads from Chromotek.
For immunoprecipitation (IP), 1 mg of cell extract was incubated with 5 µg of anti-I-A/I-E (MHC II clone M5/114, eBioscience) for 2 h at 4 °C followed by incubation with 20 µL of protein A (50% slurry, GE Healthcare) plus sepharose beads for either 2 h at room temperature or overnight at 4 °C.
In YPD, YPSuc and YPInu media, total inulinase activities were 600, 650 and 1920 U mg of cell dry weight-1 at 30°C, respectively, and 1140, 1290 and 1390 U mg of cell dry weight-1 at 45°C, respectively.
10 to 20 mg of cell walls was suspended in 1 ml of 1.6 N NaOH.
Briefly, 45 mg of cell sediment were suspended in Tris-HCl (pH 7.4) buffer containing 1% sodium dodecyl sulfate.
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