Protein phosphorylation is an important signal transduction mechanism within the cell, which is involved in many cellular functions, such as growth, death, and response. Phosphorylated proteins play a key role in cell signaling. Therefore, studying protein phosphorylation is important for understanding cell biology and disease progression.
Figure 1Acidification quantitative proteomics analysis workflow.
The assay of protein phosphorylation is typically performed using the following methods:
1. Radiolabeling method:
-32P ATP is typically used as a radiophosphate donor, catalyzed by protein kinases, to phosphorylation of the protein of interest. Then, the level of protein phosphorylation is detected by radiometric assay.
2、western blotting：
Detection is performed using antibodies that are specific to specific phosphorylation sites. First, the protein samples are separated by SDS-PAGE electrophoresis, then transferred to PVDF or Nitrocellulose membranes, and then detected with specific phospho-antibodies.
Figure 2Far-Western blot analysis.
3. Mass spectrometry:
Mass spectrometry analysis of proteins or peptides allows precise determination of the site and extent of protein phosphorylation. Commonly used techniques include liquid chromatography tandem mass spectrometry (LC-MS).
4. Phos-tag gel electrophoresis:
phos-tag is a compound that binds to phosphorylation sites. In phos-tag SDS-PAGE, phosphorylated proteins move more slowly in the gel, separating from non-phosphorylated proteins.
5. ELISA (enzyme-linked immunosorbent assay):
With phospho-specific antibodies, ELISA experiments can be constructed to determine the level of phosphorylated proteins in a sample.
6. Flow cytometer:
Phosphorus-specific antibodies and fluorescent labels allow flow cytometry to analyze protein phosphorylation at the single-cell level.
When choosing a method for protein phosphorylation assays, consider the nature of the sample, the required sensitivity and resolution, the available instrumentation, and the intended goals of the experiment.