Using proximity ligation assay to detect protein arginine methylation

Area of Science:

• Molecular Biology
• Cellular Biology
• Biochemistry

Background:

• Arginine methylation is a key post-translational modification influencing proteome diversity and cellular functions.
• Assessing endogenous protein arginine methylation in cells is increasingly important for biological research.
• Classical methods like immunoprecipitation have limitations in cellular context analysis.

Purpose of the Study:

• To detail a proximity ligation assay (PLA) protocol for detecting and quantifying protein arginine methylation in situ.
• To demonstrate the utility of PLA in conjunction with RNA interference and methyltransferase inhibitors.
• To establish a method for studying arginine methylation within a native cellular environment.

Main Methods:

• In situ proximity ligation assay (PLA) for detection, localization, and quantification of arginine methylation.
• Application of RNA interference and specific methyltransferase inhibitors.
• Automated quantification using Image J software.

Main Results:

• A standard PLA protocol was successfully applied to detect arginine methylation.
• The glucocorticoid receptor was identified as being methylated by PRMT5 in the nucleus of MCF-7 cells.
• Automated quantification of protein arginine methylation using Image J was demonstrated.

Conclusions:

• Proximity ligation assay (PLA) provides a novel and effective approach for studying protein arginine methylation in situ.
• The described PLA protocol allows for simultaneous detection, localization, and quantification of arginine methylation.
• This technique has the potential to be extended to investigate other post-translational modifications.