Abz-GFDPFRQ-EDDnp

Abz-GFDPFRQ-EDDnp (10 μM) serves as an effective fluorescent substrate for detecting TOP enzyme activity in human hippocampal tissue extracts, and its assay results show that TOP activity is decreased in the hippocampal tissues of temporal lobe epilepsy model^[1].
Experimental method for detecting TOP enzyme activity in brain tissue extracts using Abz-GFDPFRQ-EDDnp refers to ^[1]:
1. Sample pretreatment:
Homogenize human hippocampal tissue samples in lysis buffer (50 mM Tris-HCl pH 7.4, 50 mM NaCl, 0.1% Triton X-100), determine the protein concentration in the extract using the Lowry method, and set it aside for use.
2. Reagent preparation:
Prepare 50 mM Tris-HCl buffer (pH 7.4, containing 100 mM NaCl) as the reaction buffer; separately prepare Abz-GFDPFRQ-EDDnp fluorescent substrate with a final concentration of 10 μM, TOP-specific inhibitor JA-2 with a final concentration of 2 μM, neurolysin-specific inhibitor Pro-Ile with a final concentration of 0.5 mM, and 0.5 mM dithiothreitol (DTT).
3. Enzyme activation and reaction system construction:
Add an appropriate amount of hippocampal tissue extract (containing the target protein) and DTT (final concentration 0.5 mM) to the reaction system, and incubate at 37°C for 5 minutes to activate TOP enzyme; subsequently add Abz-GFDPFRQ-EDDnp substrate (final concentration 10 μM), and add Pro-Ile (to distinguish TOP from neurolysin activity) according to experimental requirements to construct a complete reaction system.
4. Reaction monitoring:
Place the reaction system in a fluorometer, continuously monitor changes in fluorescence signals at 37°C, with the excitation wavelength (λex) set to 320 nm and the emission wavelength (λem) set to 420 nm.
5. Specificity verification and result calculation:
Set up a negative control system containing JA-2 (final concentration 2 μM) (for inhibiting TOP activity), eliminate non-specific reaction interference by comparing differences in fluorescence intensity among the blank control, negative control and sample groups; TOP enzyme activity is expressed as nanomoles of substrate hydrolyzed per milligram of protein per minute (nM/min/mg), all measurements are performed in triplicate, and data are statistically analyzed using Student's t-test.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.