Methylation Analysis

Methylation analysis is an experimental method used for detection and study of DNA, RNA, and protein methylation. Protein methylation involves adding a methyl group to specific amino acid residues in a protein, typically lysine or arginine. This modification has significant effects on protein function, interactions, and localization. Below are some commonly used techniques for protein methylation analysis:

 

1. Mass Spectrometry

(1) Purpose: Mass spectrometry is the primary method for identifying and quantifying protein methylation. It can provide detailed information about methylation sites, the extent of methylation, and methylation dynamics.

(2) Process: The protein sample is digested and ionized for analysis by mass spectrometry. Software is used for data analysis to identify methylation sites.

 

2. Immunoprecipitation

(1) Purpose: Using specific antibodies to enrich methylated proteins or peptides, which can be used for subsequent mass spectrometry or Western blot.

(2) Process: Methylated proteins are captured using antibodies against specific methylation sites (e.g., specific lysine or arginine methylation antibodies).

 

3. Western Blot

(1) Purpose: Western blot is used to detect the presence and abundance of methylated proteins in a sample.

(2) Process: Protein detection is done using antibodies against methylation sites, allowing for qualitative analysis of methylation levels.

 

4. 2D Gel Electrophoresis

(1) Purpose: By separating modified and unmodified protein forms, it can observe how methylation affects protein isoelectric point and molecular weight.

(2) Process: Proteins are first separated by isoelectric point, then by molecular weight, and visualized through staining or Western blotting.

 

5. Real-Time Fluorescence Resonance Energy Transfer (FRET)

(1) Purpose: Used to study how methylation affects interactions between proteins.

(2) Process: Monitors energy transfer changes when methylation status changes using labeled proteins.