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Identical concentrations of the mutant and wild-type proteins were used to avoid any differences in the spectra arising from differences in their concentrations.
An enzyme solution was prepared containing 9 nM [α-P]ATP (5 μCi), 0.25 mM acetyl CoA, 5 mM MgCl2, 20 mM NaHCO3 in 0.1 M Tris-HCl buffer (pH 7.8), and increasing concentrations of the mutant enzymes (2.2, 4.5, 8.9, 17.8, and 35.6 μM) in a total volume of 100 μL.
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Equal concentrations of Mutant 1, Mutant 2 (Mutant 1 with reversion at position 238), Mutant 3, Mutant 4 (see Table 1), and wildtype rHRP were applied (see Methods).
In contrast, when we tested higher concentrations of the Cry1Ab-R99A mupanto up to 10 or 100 fold higher concentration of the mutant than the wildtype toxin; we found that Cry1Ab-R99A wableble to inhibit toxicity of wildtype toxin (Fig. 1B).
Deletion of arcA reverts the extracellular acetate concentration of the mutant back to wt levels.
Aggregate formation correlates with the total concentration of the mutant protein.
This could depend on the concentration of the mutant protein in the spine, as well as other unknown factors.
The quantification of the ATP level revealed that the ATP concentration of the mutant with the redesigned chitinase was also about 10-fold higher than the ATP concentration of the control strains.
The rate constants of MgATP hydrolysis at each concentration of the mutant enzymes, the Kd of MgATP binding, and the kcat of MgATP hydrolysis were determined as for the wild-type enzyme.
The simulation results showed that the mutant neuronal SSNM had similar behavior to the yeast SSNM: The fusion was amplified by increasing the concentration of nSM in the mutant nSM-SNARE system and the stimulation effect monotonically increased with the concentration of the mutant nSM protein.
Because the growth rate of S. pneumoniae is significantly slower than many other Gram-positive bacteria in culture media, the problems associated with the unstable mutation become substantial when attempting to generate a considerable concentration of the mutant.
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