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The same assay was used to monitor the stability of the enzyme upon storage and as a calibration assay for the analysis of enzymatic activity by HPLC.
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For the calibration assays, linear regression analysis was conducted by plotting response area vs. concentration.
Calibration assays using pure lactate (no cells) showed a linear response from 10 to 200 μM lactate, which is comparable to a 50 μL microtiter plate assay using the same reagents (Fig. 1b; Additional file 1: Fig. S1).
Calibration assays were performed to determine optimal ADC dosing, thus enabling the elimination of the largest possible NCAM1+ cell fraction from hFK cells without harming other cells (Supporting Information Fig S5D and E).
The results obtained with various assays show wide variability due to differences in the antibodies used and the distinct reference preparations employed for the calibration of assay kits.
After calibration with Assay Buffer, the bead mixture was gently mixed with a pipette and sonicated for 10 s and then 25 μl 1 × phosphoprotein beads was added to each well.
Furthermore, our results suggest that a simple reliable calibration between assays cannot be accomplished.
A validation chromatographic run included a set of calibration samples assayed in duplicate and quality control samples at four levels in triplicate.
Calibration of assays with standards (for example, recombinant proteins or reference standards prepared from large pools of patient samples) can help to improve consistency and reproducibility across assay runs.
Titres were calibrated to ensure comparability with other published work using the same assay (calibration factors provided by Mark Esser, personal communication).
For validation, matrix-matched calibration and recovery assay were applied.
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