Common Methods of Protein Phosphorylation Modifications Detection

Protein phosphorylation modification is a key event in cellular signal transduction, therefore accurate detection and quantification of it are essential. The commonly used methods for detecting protein phosphorylation modification mainly include:

 

1. Western Blot

Use antibodies specific to the phosphorylation site to detect phosphorylated proteins. This method can provide semi-quantitative data for specific phosphorylation sites.

(1) Advantage: relatively simple, sensitive; can perform qualitative and quantitative analysis.

(2) Disadvantage: requires highly specific antibodies; cannot confirm the precise phosphorylation site.

 

2. Immunoprecipitation (IP)

First, use a specific antibody to precipitate the target protein from the cell lysate, then perform Western Blot to detect its phosphorylation situation.

(1) Advantage: can study protein-protein interactions.

(2) Disadvantage: requires high-quality antibodies; may lose some weak interactions.

 

3. Mass Spectrometry (MS)

It is particularly suitable for identifying new phosphorylation sites and performing quantitative analysis of phosphorylated proteins. Combined with liquid chromatography (LC-MS/MS), it can identify and quantify thousands of phosphorylation sites.

(1) Advantage: can determine the precise phosphorylation site; strong quantitative ability; can identify multiple phosphorylation sites.

(2) Disadvantage: the equipment is expensive and requires professional knowledge; there are many sample handling steps.

 

4. Protein Phosphorylation Arrays

This is a high-throughput method that can detect the phosphorylation status of multiple proteins in a single experiment.

(1) Advantage: high throughput; can target multiple targets simultaneously.

(2) Disadvantage: may require complex sample handling and data analysis.

 

5. Radiolabeling

For example, labeled cells with [^32P] sodium orthophosphate, then perform immunoprecipitation and electrophoresis on the protein, and detect its radioactivity to determine the phosphorylation state of the protein.

(1) Advantage: extremely high sensitivity and direct detection.

(2) Disadvantage: involves radioactive materials, health and safety risks, and cannot determine the specific phosphorylation site.

 

6. Flow Cytometry

Use specific phosphorylation antibodies and fluorescently labeled secondary antibodies, can detect phosphorylation events at the single cell level.

(1) Advantage: can analyze at the single cell level; can be combined with other cell markers for multiparameter analysis.

(2) Disadvantage: requires specific antibodies; only applicable to suspensible cell samples.

 

7. ELISA (Enzyme-Linked Immunosorbent Assay)

Using specific phosphorylation antibodies, can perform quantitative analysis of phosphorylated proteins.

(1) Advantage: suitable for high-throughput screening; accurate quantification.

(2) Disadvantage: requires specific antibodies; can only test for known targets.

 

Each method has its advantages and limitations, and the choice of method depends on the purpose of the experiment, the available equipment, and the type of sample.