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However, in our loading assay, the loaded scnRNAs were detected by immuno-precipitating Twi1p, and thus, we most likely observed the only end point of the loading process.
The static loading assay determined the deformations, through strain gages (single-element gage, two-element gage and three-element rosettes), and the displacements along the sleeper, using linear displacement transducers.
On the contrary, during a loading assay, the fraction of bound peptide at the steady state depends on peptide and DR concentration, as predicted.
Figures 1B, b (TRAP-gel loading assay), c (Fluorometric TRAP assay) show the enzymatic activity of telomerase in lysates from 12, 24, 48 and 72 hours post fertilization (hpf) embryos.
In the loading assay above, the cell lysate was not supplied with ATP.
(B ) The dye loading assay demonstrates gain of CO2-sensitivity in mCx31.
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The 'catalytic rate enhancement' coefficient was determined in loading assays with 100 nM soluble HLA-DR1 and 50 µg/ml HA306-318 wititratedted amounts of the catalytic peptide-MLE (1 h, 37°C, pH 7.4).
These results suggest that RNAs co-precipitated with Twi1p in the cell lysate-based scnRNA loading assays were properly loaded into Twi1p.
The scnRNA loading assays above detected the interaction between Twi1p and the 27-nt RNAs but did not show that these RNAs were properly loaded into Twi1p.
First, to test whether Giw1p inhibits the loading of scnRNA duplexes into Twi1p, we performed scnRNA loading assays using cell lysates from wild-type and GIW1 KO cells.
Next, to understand the relationship between the ATP-independent loading-promoting activity of Coi12p and Giw1p, scnRNA loading assays were performed with the GIW1 KO cell lysate without ATP supplementation and with or without immunodepletion of Coi12p (Fig 7B).
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