Rhodamine-N3 chloride is a rhodamine fluorescent dye with an azide group and a click chemistry reagent (Ex/Em = 544/576 nm) capable of labeling biomolecules containing alkyne, DBCO, or BCN groups.

In situ labeling sample processing based on the gel-based ABPP method [2]
1. Thaw cells on ice. Add 200 μL cold DPBS and protease inhibitor to each sample. Lysate cells using a probe sonicator and centrifuge at 100,000 × g for 45 minutes to separate.
2. Take the supernatant (soluble proteome) and resuspend it in 200 μL cold DPBS with protease inhibitor. Standardize the protein concentration of each sample to 1 mg/mL, resulting in a volume of 43 μL.
3. Perform click chemistry on each sample using a solution containing final concentrations of 25 μM Rhodamine-N3 chloride, 1 mM TCEP, 100 μM TBTA, and 1 mM CuSO₄, to a final volume of 50 μL. Incubate at room temperature for 1 hour.
4. Add 20 μL of 4X SDS loading buffer to each sample, load 30 μL of sample, and perform electrophoresis on a 10% SDS-PAGE gel. Image using a fluorescence scanner.

DMSO: 20 mg/mL (23.6 mM), Sonication is recommended.