Rate assay of with 4-nitrophenyl as an artificial substrate

Synthesis and Conformational Analysis of Cyclotetrapeptide Mimetic β‐Turn Templates and Validation as 3D Scaffolds

“…This product is a chromogenic substrate for β‐ N ‐acetylglucosaminidase that is transformed in 4‐nitrophenol, the absorbance of which is measured at λ =405 nm. As previously described,29 there is a linear correlation between absorbance and enzymatic activity. It is therefore possible to identify the number of adherent cells in treated wells, interpolating the absorbance values of the unknowns in a calibration curve.…”

Effects of a High-Pressure Treatment on Bovine Gamma Globulin and Its Reduction in Allergenicity

“…Fifty microliters per well of a BGG solution (5 mg/ml in a 50 mM sodium carbonate buffer at pH 9.6) was added to a 96-well ELISA plate. The plate was incubated at 30 C for 2 h, washed five times with phosphate-buffered saline (PBS, 137 mM NaCl, 8 mM Na 2 HPO 4 . 12H 2 O, 2.7 mM KCl and 1.5 mM KH 2 PO 4 at pH 7.4), blocked at 30 C for 1 h with a 2% gelatin-PBS solution, and then washed five times with 0.05% Tween20 in phosphate-buffered saline (PBST, pH 7.4).…”

“…12H 2 O, 2.7 mM KCl and 1.5 mM KH 2 PO 4 at pH 7.4), blocked at 30 C for 1 h with a 2% gelatin-PBS solution, and then washed five times with 0.05% Tween20 in phosphate-buffered saline (PBST, pH 7.4). Fifty microliters of serum (diluted 20-fold in PBST) was then added to the plate which was incubated at 30 C for 1 h. The plate was washed five times with PBST, and then the second antibody (an anti-human IgE alkaline phosphatase conjugate; Biosource, San Jose, CA, USA) was added. The plate was again washed five times with PBST.…”

“…The plate was again washed five times with PBST. One hundred microliters of p-nitrophenyl phosphate (10 g in a 10% diethanolamine buffer at pH 9.8, 10 ml) was added, and after the plate had been incubated at 30 C for 60 min, the absorbance was measured by a microplate reader with a 405-nm filter.…”

“…Stimulation was stopped by placing the cells on ice. After centrifugation, the amount of -hexosaminidase activity released from the cells in the supernatant was measured according to the method of Shibata et al 30) and Matsuda et al 31) with some modifications. Fifty microliters of supernatant and 50 ml of 1 mM 4-nitrophenyl N-acetyl--D-glucosaminide (in a 0.1 M sodium citrate buffer at pH 4.5) were mixed in a 96-well plate, and then incubated at 37 C for 2 h. The reaction was stopped by adding 200 ml of a 0.1 M sodium bicarbonate buffer (pH 10.0), and the -hexosaminidase activity in the supernatant was quantified by measuring the absorbance with a microplate reader having a 405-nm filter.…”