CGTDP

Guidelines
Guidelines (The following is a recommended experimental protocol for guidance only, and should be adjusted according to your specific requirements)^[1].

1. Stock Solution Preparation
1.1 Solvent: DMSO.
1.2 Concentration recommendation: 4 mM.

2. Working Solution Preparation
2.1 Working concentration: Take 2 mL of 5 μM creatine kinase (CK) solution (solvent: 0.1 M sodium bicarbonate, pH 8.6), and add 100 μL of 4 mM CGTDP dissolved in DMSO.
2.3 Precautions: Adjust the working solution concentration as needed; prepare and use immediately.

3. Staining Procedure
3.1 Sample type: Protein sample (rabbit creatine kinase, CK).
3.2 Incubation conditions: Incubate CK (5 μM) with the CGTDP working solution at 30 °C for 1.5 h.
3.3 Washing steps: Separate the labeled protein from excess reagents using a Sephadex G-25 column with 0.1 M NaHCO₃ (pH 8.6) as the eluent; dialyze the collected protein fractions in 0.03 M NH₄HCO₃ solution.

4. Control Group Setup
4.1 Perform the same labeling procedure on CK alone under conditions without CGTDP.

5. Detection and Analysis
5.1 Instrument: Fluorimeter
5.1.1 Excitation wavelength: 493 nm; emission wavelength of CGTDP-labeled CK: 590 nm.
5.2 Result analysis:
5.2.1 Change in fluorescence intensity: The fluorescence quantum yield of CGTDP-labeled CK increases by approximately 4-fold compared with free CGTDP.
5.2.2 Fluorescence localization: CGTDP labels the arginine residue at the active site of CK.
5.2.3 Fluorescence shift: The maximum emission wavelength blue-shifts from 607 nm of free CGTDP to 590 nm of CGTDP-labeled CK, indicating that the active site of CK is in a relatively hydrophobic environment; acid denaturation or heat denaturation of the labeled CK causes a further blue-shift of the probe's maximum emission wavelength, reflecting conformational changes of the active site and a decrease in local polarity.