Sortase A Activity Assay[1-2]
This plan is based on literature, please modify it to meet your specific needs.
1. Preparation of substrate working solution
(1) Prepare storage solution: Dissolve Bacterial Sortase Substrate III, Abz/DNP (TFA) in DMSO at concentration of 10mM.
Note: Unused storage solution should be aliquoted and stored in the dark at -20°C or -80°C to avoid repeated freezing and thawing.
(2) Prepare working solution: Dilute the stock solution with reaction buffer (50mM Tris-HCl, 150mM NaCl; pH=7.5).
Note: Prepare the working solution fresh and store briefly at 4°C or on ice.
2. Prepare a 96-well plate, including:
Experimental wells: a mixture of Bacterial Sortase Substrate III, Abz/DNP (TFA) substrate and Sortase A, concentrations of 10μM and 5-15μM, respectively.
Sample blank wells: Bacterial Sortase Substrate III, Abz/DNP (TFA) substrate only, concentration 10μM, add reaction buffer to make up the volume.
Substrate blank wells: Sortase A only, concentration 5-15μM, add reaction buffer to make up the volume.
4. Incubate the 96-well plate at 37℃ for 10-60 min, and detect the fluorescence intensity at 320nm under 420nm excitation using a microplate reader.
Precautions:
1) Fluorescent substrate has quenching problems. Please avoid light as much as possible to slow down fluorescence quenching.
2) For your safety and health, please wear a lab coat and disposable gloves.
References:
[1] Gaspar AH, Marraffini LA, Glass EM, Debord KL, Ton-That H, Schneewind O. Bacillus anthracis sortase A (SrtA) anchors LPXTG motif-containing surface proteins to the cell wall envelope. J Bacteriol. 2005;187(13):4646-4655.
[2] Chen Y, Li W, Wang L, Wang B, Suo J. Novel inhibition of Staphylococcus aureus sortase A by plantamajoside: implications for controlling multidrug-resistant infections. Appl Environ Microbiol. 2025;91(1):e0180424. |
