P2-Aggregate

Operating Instructions
(The following is a recommended experimental protocol for guidance only, and adjustments are required based on your specific needs)

1. Stock Solution Preparation
1.2 Concentration recommendation: High-concentration stock solution of 1-10 mM.

2. Working Solution Preparation
2.2 Working concentration: 5 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare and use immediately.

3. Staining Procedure
3.1 Sample type: Adherent cells (HEK293T cells)^[1].
3.1.1 Adherent cells: Trypsin digestion is not required as specified for imaging experiments.
3.2 Incubation condition: Incubate in the dark.

4. Control Setup
4.2 Set up negative and positive controls:
4.2.1 Positive control: HEK293T cells expressing aggregated Htt-110Q-Halo or aggregated SOD1 (G85R)-Halo, treated with the proteasome inhibitor MG132.
4.2.2 Negative control: HEK293T cells expressing folded Htt-25Q-Halo or SOD1 (G85R)-Halo, without treatment with the proteasome inhibitor MG132 (HY-13259).

5. Detection and Analysis
5.1 Instrument: Confocal fluorescence microscope; Ex wavelength = 543 nm.
5.1.1 Excitation/emission wavelength: Ex = 543 nm; the emission wavelength correlates with local polarity (the lower the polarity, the shorter the emission wavelength).
5.2 Result analysis:
5.2.1 Fluorescence intensity change: Fluorescence is retained in both polar (folded protein) and non-polar (aggregated protein) environments, which can be used to identify transfected cells.
5.2.2 Fluorescence localization: The signal shows a diffuse pattern in cells expressing folded proteins; the signal shows a punctate pattern in cells expressing aggregated proteins; the punctate structures exhibit polarity heterogeneity (the polarity of the outer shell is higher than that of the inner core).
5.2.3 Color change: Compared with folded proteins, aggregated proteins have reduced polarity, and their emission wavelength undergoes a blue shift.
5.2.4 Quantitative analysis: Calculate the local polarity using the linear correlation between emission wavelength and dielectric constant (y = 19.43ln (x) + 581.95, R² = 0.90).