Sortase A activity was measured by a fluorescence resonance energy transfer method as reported previously with some modifications.10 (link) The assay was performed in the wells of a 96-well black plate. The synthetic fluorescent peptide Dabcyl-QALPETGEE-Edan (GL Biochem Ltd., Shanghai, China) was used as a model substrate. Recombined SrtAΔN59 was purified from E. coli and the enzyme purity was examined by SDS-PAGE. Briefly, different concentrations of CHQA were added to the reaction buffer (50 mM Tris·HCl, 150 mM NaCl, 5 mM CaCl2, pH 7.5) containing 10 μM SrtAΔN59 in a final volume of 300 μL. The plate was incubated at 37 °C for 30 min. Subsequently, 10 μM of substrate peptide was added and keep a further incubation at 37 °C for 1 h. The fluorescence intensity was measured at 495 nm with an excitation wavelength of 350 nm using a microplate reader (PE envision, PerkinElmer Co., Waltham, USA), and the fluorescence changes were used to calculate the inhibitory rates.
Pe envision
Manufactured by PerkinElmer
Sourced in United States
The PE Envision is a microplate reader designed for high-throughput screening and quantitative analysis of biomolecular interactions. It offers a compact and versatile platform for a range of applications, including cell-based assays, ELISA, and fluorescence-based detection.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using pe envision
1
Sortase A Inhibition Assay Protocol
2
Quantifying Intracellular and Extracellular ATP in S. aureus
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The intracellular and extracellular ATP concentrations of S. aureus were determined according to the method described previously with some modifications.4 (link) Briefly, logarithmic phase S. aureus cells were harvested by centrifugation at 3000 rpm for 5 min, washed twice and resuspended in 0.85% sterile saline at a cell density of 1 × 109 CFU mL−1. The bacterial suspensions were treated with different concentrations of CHQA at 37 °C for 30 min. Then, the samples were centrifuged at 5000 rpm for 5 min, the supernatants and cell pellets were stored on ice respectively to prevent ATP loss. The ATP concentration of the supernatants, which represents the extracellular concentration, was determined using an ATP assay kit (Life Technologies, Eugene, OR, USA) following manual instructions with a microplate reader (PE envision, Perkin Elmer Co., Waltham, MA, USA). To measure the intracellular ATP concentration, the cell pellets were washed, resuspended in 0.85% sterile saline, and treated by ultrasound on ice. After centrifugation at 10 000 rpm for 3 min, the ATP concentration of the resultant supernatants, which represents the intracellular concentration, was determined as described for the extracellular ATP concentration.
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