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Various site-specific modification reactions were performed on biological mCRP to block or alter selected amino acid R groups prior to performing binding assays.
Both modification reactions were confirmed by FTIR and 1H NMR, obtaining a degree of substitution (DS) of 31% and 26% for Cs Fu and Cs AMI, respectively.
The modification reactions were found to occur without alteration of the original enantiomeric composition but with a significant reduction in molecular weight.
DMS modification reactions were stopped by addition of 59.3 µℓ of DMS stop buffer (1 M Tris-HCl pH 7.5, 0.1 M EDTA pH 8.0, and 1 M 2-mercaptoethanol).
CMCT modification reactions were then stopped by addition of 300 µL stop buffer (0.3 M sodium acetate, 0.2 M PIPES pH 6.5, and 5 mM EDTA) and placed on ice for 3 minutes.
Modification reactions were examined by mass spectrometry, and residues labeled by six different agents were determined.
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The different enzyme-driven modification reactions are specific to the compartments of the Golgi apparatus.
As is usual the case [S]» [E0] and that the modification reactions are relatively slow compared with the set up of the steady-state of the enzymatic reaction.
The progress of the modification reactions was confirmed by elemental analysis, which allowed calculation of the surface coverage density with bonded ligands.
The enzymes involved in these modification reactions are either LanM enzymes, that catalyse both reactions (dehydration of the hydroxy amino acids and cyclization), or a combination of a LanB enzyme (dehydration of Ser and Thr residues) and a LanC enzyme (thioether formation) is present.
Quantification of chemical modification reactions was performed using the Fuji-FLA-5100 Fuji-FLA-5100 Fuji-FLA-5100the AIDA software package according to the manufacturers procedures.
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