TNO211

Guidelines (The following is our recommended experimental protocol. This protocol is for guidance purposes only and needs to be adjusted according to your actual requirements).

1. Stock Solution Preparation
1.1 Solvent: anhydrous DMSO.

2. Working Solution Preparation
2.1 Diluent: Buffer A (50 mM Tris (pH 7.6), 150 mM NaCl, 5 mM CaCl₂, 1 μM ZnCl₂, 0.01% Brij 35).
2.2 Working concentration: 1-5 μM.
2.3 Notes: Adjust the working solution concentration according to actual requirements; prepare fresh before use.

3. Staining Procedure
3.1 Sample types^[1]:
3.1.1 Synovial fluid samples: Centrifuge at 10000×g for 15 min at 4°C, then dilute 5-fold with Buffer A.
3.1.2 Cell culture medium samples: Take 75 μL of serum-free conditioned medium obtained from cultured human umbilical vein endothelial cells.

3.2 Incubation conditions: Pre-incubate the enzyme and TNO211 separately at 37°C for 5 min, then incubate at 37°C for up to 6 h according to experimental requirements.
3.3 Incubate 19 kDa MMP-1, MMP-2, MMP-3, or MMP-9 with TNO211 at 37°C, remove 50 μL aliquots at set time points, and add an equal volume of pre-cooled 40 mM EDTA (dissolved in 0.1% TFA) to terminate the reaction.

4. Detection and Analysis
5.1 Instrument: A fluorescence spectrophotometer equipped with an appropriate detection module.
5.1.1 Excitation/emission wavelengths: Set the excitation wavelength for fluorescence measurement to 340 nm and the emission wavelength to 485 nm.

5.2 Result analysis:
5.2.1 Intact TNO211 exhibits almost no fluorescence signal, because the EDANS fluorophore is quenched by the Dabcyl group to a degree of 98.4%.
5.2.2 TNO211 is specifically cleaved by MMPs at the Gly-Leu bond, generating two different fragments: a fragment containing the N-terminal Dabcyl and a fluorescent fragment containing the C-terminal EDANS.
5.2.3 The fluorescence intensity of the fragment containing the C-terminal EDANS is proportional to the amount of substrate cleaved, and the rate of fluorescence increase is linearly correlated with the concentration of active MMP.