Protein Translation and Post-Translational Modification Detection Methods

Post-translational modifications (PTMs) are key biochemical processes in cells that modify the chemical properties, structure, function, and location of proteins. Common post-modification of proteins includes phosphorylation, acetylation, ubiquitination, glycosylation, pantothenylation, etc. Detecting these modifications is crucial for understanding the role and regulatory mechanisms of proteins. Below are the commonly used methods to detect protein post-translational modifications:

 

1. Western Blot

Uses antibodies that are sensitive to specific modifications to detect specific protein modifications. For example, antibodies specific to phosphorylation sites are used to detect the phosphorylation status of proteins.

 

2. Co-Immunoprecipitation (Co-IP) and Immunoprecipitation (IP)

Used to isolate and enrich proteins with specific modifications from complex cell lysates.

 

3. Mass Spectrometry (MS)

Currently the most powerful tool for detecting and quantifying protein post-translational modifications. It can accurately identify the modification sites and types of proteins.

 

4. Radiolabeling

For instance, in phosphorylation studies, radioactive γ-32P ATP can be used to label phosphorylated proteins.

 

5. Enzyme-Linked Immunosorbent Assay (ELISA)

Uses specific antibodies to detect and quantify specific protein modifications.

 

6. Fluorescence Resonance Energy Transfer (FRET)

Used to monitor protein modifications in real-time in live cells.

 

7. Immunofluorescence Microscopy

Can be used to locate specific protein modifications in cells or tissue sections.

 

8. Flow Cytometry (FACS)

Can be used to detect and quantify specific protein modifications on the cell surface or inside cells.