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Preparation and stability of solutions: APMA-solution: 40 mM p-Neutrophenyl mercuric acetate (APMA) in dimethylsulfoxide. The solution is stored at -20 °C. Peptide hydrolysis buffer: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl 2 , 0.025 % Brij 35. The solution is stable for several weeks at 4 °C. Stock solution of peptide substrate: 100 µM solution of Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg in 20 % dimethylsulfoxide. The solution is stored at -20 °C. Stock solution of unquenched peptide: 10 µM solution of (7-methoxycoumarin-4-yl) acetyl-Pro-Leu-NH 2 (Mca-Pro-Leu) in 20 % dimethylsulfoxide. The solution is stored at -20 °C. Activation: An aliquot of 19.5 µL Neutrophil procollagenase is mixed with 0.5 µL APMA solution and the mixture is incubated for 30 min at 37 °C.
The activity of Neutrophil procollagenase is measured fluorimetrically with a synthetic internally quenched fluorescent substrate according to Knight et al. An excitation wavelength of 328 nm and an emission wavelength of 393 nm are set in an appropriate fluorimeter. The instrument is calibrated with the unquenched peptide Mca-Pro-Leu at a concentration corresponding to between 2 and 10 % hydrolysis of the protease substrate. Kinetic reactions are conveniently carried out in a constant volume of 2.5 mL . The substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg is diluted in peptide hydrolysis buffer to a concentration of 0.8 µM and equilibrated at a temperature of 37 °C. Aliquots of 1 µL to 2 µL of the activation mixture are than added and the increase in fluorescence is recorded over a time interval between 2 and 12 min. Activity units per mL enzyme solution are calculated according to the following equation: c Mca-Pro-Leu deltaF Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg Activity U/mL = ?????.. V total F Mca-Pro-Leu v enzyme c Mca-Pro-Leu : Concentration of Mca-Pro-Leu used for calibration of the fluorimeter ( umoles/mL) F Mca-Pro-Leu : Fluorescence of Mca-Pro-Leu at the concentration c Mca-Pro-Leu used for fluorimeter calibration deltaF Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg : Change in fluorescence during peptide hydrolysis per min V : Volume of peptide hydrolysis reaction (2.5 mL ) v : Volume of added enzyme (0.001 mL to 0.002 mL ) 5. References.