A robust method for particulate detection of a genetic tag for 3D electron microscopy

Area of Science:

• Cell Biology
• Biochemistry
• Microscopy

Background:

• Genetic tags are crucial for protein localization in cells.
• Ascorbate peroxidase (APEX) is a versatile genetic tag for electron microscopy (EM).
• Current EM methods can have limitations in signal-to-noise ratio and quantification.

Purpose of the Study:

• To develop a method (APEX-Gold) to enhance APEX tag detection in EM.
• To improve signal-to-noise ratio and enable quantification of tagged proteins.
• To demonstrate the broad applicability and sensitivity of the APEX-Gold method.

Main Methods:

• APEX-Gold converts the APEX reaction product into silver/gold particles.
• The method was tested with various protein types (membrane, cytoplasmic, cytoskeletal).
• Compatibility with advanced EM techniques (cryo-EM, FIB-SEM, tomography) was assessed.

Main Results:

• APEX-Gold significantly increases signal-to-noise ratio for EM detection.
• The method provides a quantifiable particulate signal for tagged proteins.
• APEX-Gold demonstrated high sensitivity, detecting low-level fusion proteins in CRISPR-edited mouse cells.

Conclusions:

• APEX-Gold offers unambiguous and quantifiable detection of APEX-tagged proteins via EM.
• The method is versatile, compatible with multiple EM techniques, and highly sensitive.
• APEX-Gold facilitates accurate quantitation of protein expression levels.