HBC (HBC 530)

HBC530-based analysis of metalloproteinase 2 (MMP-2)

This protocol only provides a guideline, and should be modified according to your specific needs.

1. Preparation of HBC530 solution

2μM HBC530 working solution was prepared from 1mM HBC530 stock solution (dissolved in DMSO). NOTE: The working solution must be prepared fresh on the day of the experiment.

2. Imaging of MMP-2 by HBC530 in the extracellular matrix (ECM)

(1) For imaging of MMP-2 in the ECM, the HePG2 cells were pretreated with TGF-β for 24h.

(2) The 10μl of 100μM protein-nucleic acid chimeric probe (PNCP) was first prepared in the reaction buffer (50mM HEPES, 50mM NaCl, 10μM MnCl₂, and 1mM MgCl₂) at 37°C for 6h, and then 100μl of the transcription buffer containing 1μM PNCP and 4mM rNTP was added to the cell culture dish for 2h.

(3) 2μM HBC530 working solution was incubated with HepG2 for 15min. A microplate reader was used to record the fluorescence signals.

3. Caveats.

(1) Dark operation: HBC530 working solution has a half-life of 4h at 4°C, and is prepared and used on demand.

(2) During the entire experiment, make sure to wear protective gloves for proper safety precautions.

(3) The reagents used in this experiment are for scientific research purposes only.

References:
[1] Xie X, Sun Y, Peng J, et al. Collagen anchoring protein-nucleic acid chimeric probe for in situ in vivo mapping of a tumor-specific protease[J]. Analytical Chemistry, 2023, 95(50): 18487-18496.