Protein phosphorylation modifications are a critical event in cell signaling, so accurate detection and quantification of them is critical. The commonly used methods for the detection of protein phosphorylation modifications mainly include:
1.western blot：
Phosphorylated proteins are detected using antibodies specific to the phosphorylation site. This method can provide semi-quantitative data for specific phosphorylation sites.
1.Advantages: relatively simple and sensitive; Can be analyzed qualitatively and quantitatively. 2.Disadvantages: Requires highly specific antibodies; The precise phosphorylation site cannot be determined. 2.Immunoprecipitation (IP):
Specific antibodies are used to precipitate the protein of interest from the cell lysate, and then Western blot is performed to detect phosphorylation.
1.Advantages: Protein-protein interactions can be studied. 2.Disadvantages: High-quality antibodies are required; Some weak interactions may be lost. 3.Mass Spectrometry (MS):
Ideal for the identification of new phosphorylation sites and for the quantification of phosphorylated proteins. In combination with liquid chromatography (LC-MS), thousands of phosphorylation sites can be identified and quantified.
1.Advantages: Ability to determine precise phosphorylation sites; Strong quantitative ability; Multiple phosphorylation sites can be identified. 2.Disadvantages: The equipment is expensive and requires expertise; There are many steps in sample processing4.Protein phosphorylation arrays:
This is a high-throughput method that can detect the phosphorylation status of multiple proteins in a single experiment.
1.Advantages: High throughput; It is possible to detect several targets at the same time. 2.Disadvantages: Complex sample handling and data analysis may be required. 5.Radiolabeling:
For example, cell labeling is performed using [32p]sodium orthophosphate, followed by immunoprecipitation and electrophoresis of the protein to detect its radioactivity to determine the phosphorylation status of the protein.
1.Advantages: Extremely high sensitivity and direct detection. 2.Disadvantages: Radioactive material is involved, there are health and safety risks, and the specific phosphorylation site cannot be determined. 6.Flow cytometry:
Using specific phospho-antibodies versus fluorescently conjugated secondary antibodies, phosphorylation events can be detected at the single-cell level.
1.Advantages: Ability to analyze at the single-cell level; Multiparametric analysis can be performed in combination with other cell markers. 2.Disadvantages: Requires specific antibodies; Limited to cell samples that can be suspended. 8.ELISA (enzyme-linked immunosorbent assay):
Using specific phosphoantibodies, the quantification of phosphorylated proteins can be performed.
1.Advantages: suitable for high-throughput screening; Quantitatively accurate. 2.Disadvantages: Requires specific antibodies; Testing can only be done against known targets. Each method has its advantages and limitations, and which method to choose depends on the purpose of the experiment, the equipment available, and the type of sample.