Determination of α-D-1,4-Glucan Polymerization Degree by CCVJ Method
This protocol is adapted from research data and provided for reference only. Adjustments may be necessary based on specific experimental requirements.
(1) Reagent and Sample Preparation: Dissolve CCVJ in dimethyl sulfoxide (DMSO) to prepare a 1mmol/L stock solution, and store protected from light. Dilute to the working concentration (e.g., 19µM) using sodium phosphate buffer (5mM, pH=7.0) before use. Prepare a series of solutions or dispersions of linear α-D-1,4-glucan model molecules (MM) with different degrees of polymerization (DP). The concentration range should be 1-5g/L, covering molecular weights from 0.18kDa to 70.00kDa (corresponding to DP1 to DP450).
(2) Sample Incubation and Centrifugation: Place 1.0-5.0mg of the MM sample into a 1.5mL centrifuge tube. Add 990µL of sodium phosphate buffer and 10µL of the CCVJ stock solution to achieve a final CCVJ concentration of 19µM. Vortex to mix thoroughly, then incubate at 100°C for 1 hour. After cooling to room temperature, centrifuge at 8000g for 15 minutes. Collect the supernatant and let it stand protected from light for 1 hour before use.
(3) Fluorescence Spectrum Measurement: Transfer 150µL of the supernatant to a micro quartz cuvette. Use a fluorescence spectrophotometer to scan the CCVJ fluorescence spectrum with an excitation wavelength of 420nm and an emission wavelength range of 450-600nm (or monitor specifically around 491nm). Set the slit width to 10 nm and the temperature to 25°C. Record the fluorescence intensity at the maximum emission wavelength (approximately 491-494nm).
Precautions:
(1) CCVJ is light-sensitive. All operations should be performed with protection from light. Temperature and pH conditions during incubation and measurement must be strictly controlled.
(2) For your safety and health, please wear a lab coat and disposable gloves during operation.
References:
[1] Huang M, Guo S, Li Z, et al. Molecular rotor as an in-situ fluorescent probe for the degree of polymerization of α-D-1,4-glucans. Carbohydr Polym. 2024 Jan 15;324:121573. |
