Detection and characterization of protein aggregates by fluorescence microscopy

Area of Science:

• Biochemistry
• Pharmaceutical Science
• Analytical Chemistry

Background:

• Protein aggregation can negatively impact drug stability and biological activity.
• Accurate detection of protein aggregates is essential for protein characterization and pharmaceutical formulation development.

Purpose of the Study:

• To present a novel technique for detecting and characterizing protein aggregates using Nile Red fluorescence microscopy.
• To evaluate the sensitivity and applicability of Nile Red staining for various protein samples, including highly concentrated ones.

Main Methods:

• Protein aggregates were analyzed using fluorescence microscopy after staining with the hydrophobic probe Nile Red.
• Nile Red staining was compared with Congo Red and Thioflavine T staining.
• Aggregate diameter and number were determined and correlated with light scattering measurements.

Main Results:

• Nile Red staining enabled detection and characterization of protein aggregates, even in highly concentrated samples (193 mg/ml), without sample modification.
• The technique proved highly sensitive for detecting immunoglobulin and human calcitonin aggregates.
• Nile Red and Thioflavine T fluorescence showed colocalization, and aggregate characteristics were correlated with light scattering data.

Conclusions:

• Nile Red staining is a sensitive and effective method for detecting low levels of protein aggregates.
• This technique facilitates a deeper understanding of protein aggregation mechanisms, aiding in drug development and characterization.