Detection of Protein Phosphorylation Levels at Specific Sites

Quantitative analysis of phosphorylation levels plays a crucial role in the study of cell signaling, disease progression, and drug action mechanisms. The phosphorylation level at a specific site reflects the active state of a protein and the biological processes it participates in. Here are some methods for detecting the phosphorylation level of a protein at a certain site:

 

1. Western Blotting

The phosphorylation level of proteins can be quantified using specific antibodies.

(1) Extract the protein samples and perform SDS-PAGE electrophoresis.

(2) Transfer the proteins to the membrane.

(3) Incubate with an antibody against a specific phosphorylation site.

(4) Quantify using a second antibody labeled with fluorescence or chemiluminescence.

(5) Normalize with the signal from total protein or carrier protein to get the relative quantitative value of phosphorylation level.

 

2. Mass Spectrometry

LC-MS/MS can provide absolute or relative quantification of protein phosphorylation sites.

(1) Extract and enzymatically digest the protein samples.

(2) Enrich phosphorylated peptides through chromatography methods.

(3) Perform LC-MS/MS analysis.

(4) Use specific quantitative strategies (such as TMT, iTRAQ, or label-free methods) to obtain quantitative information on phosphorylated peptides.

(5) Compare with the non-phosphorylated control group to obtain relative or absolute phosphorylation levels.

 

3. ELISA

ELISA kits can specifically quantify the phosphorylation level at a specific site.

(1) Fix the target protein or peptide on the ELISA plate.

(2) Incubate with an antibody against a specific phosphorylation site.

(3) Use an enzyme-labeled secondary antibody and add a substrate for color development.

(4) Read the absorbance values with an enzyme marker and compare with the standard curve to get the quantitative value of protein phosphorylation level.

 

Detecting the phosphorylation level of a protein at a certain site is a key step in understanding cell function and signal transduction. Different experimental purposes and sample types may determine the most suitable quantitative method. For accurate and repeatable quantification of phosphorylation levels, choosing the appropriate experimental design and technical platform is crucial.