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The specificity of the newly developed substrates for Staphylococcus aureus V8 protease-catalyzed reactions have been examined by steady-state hydrolysis kinetic studies.
Started in 1989 to exploit research done by Richard Siegel, then at Argonne National Laboratory, outside Chicago, and Horst Hahn of the University of Saarland in Saarbrücken, Germany, the company developed substrates that eventually became useful in cosmetic applications, particularly sunscreen.
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The results clarify the newly developed substrate mimetics as efficient acyl donors as well as BL-GSE as an attractive alternative to V8 protease for enzymatic peptide synthesis.
Since AtCRT1a and CRT1b do not seem to be predominantly involved in the folding of the EFR (Figures 9; [27]), it is apparent that the different subgroups of CRTs have developed substrate specificity.
Further work that develops substrates for other KDACs and further optimizes the substrates, through for example the inclusion of non-natural amino acids, will provide the molecular reagents that may have broad value in advancing the biology of acetylation.
After a final wash in PBS/T, p-nitrophenylphosphate (Sigma-Aldrich, UK) diluted in diethanolamine buffer (Thermal Scientific, UK) was used as a developing substrate.
Tetramethyl benzidine (TMB; Pierce, Rockford, IL) was the color developing substrate and the readings were done at 450 nm.
Plates were washed and developing substrate (Mabtech) was added.
Finally, the membranes were incubated for 30 min in Sigma-Fast 3,3′-diaminobenzidine developing substrate (Sigma-Aldrich) and then rinsed in distilled water.
To further demonstrate the feasibility of applying Hsp104 to reversing disease in humans, it will be essential to develop substrate-optimized Hsp104 variants to avoid off-target effects.
Diaminobenzidine (DAB Peroxidase Substrate Kit, Vector Laboratories, Burlingame, VT, USA) was used as the developing substrate leading to a brownish color of the immunopositive cells.
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