How to Extract Phosphorylated Proteins
The extraction of phosphorylated proteins involves carefully optimized procedures and reagents to prevent dephosphorylation and ensure the integrity of labile phosphorylation sites. Below is a stepwise protocol widely adopted for efficient isolation and enrichment of phosphorylated proteins:
1. Preparation of Phosphoprotein Extraction Buffer
The extraction buffer typically contains appropriate concentrations of salts, non-ionic detergents, phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate), and protease inhibitors (e.g., PMSF, aprotinin, leupeptin). These components are essential to preserve phosphorylation states and minimize proteolytic degradation during extraction.
2. Cell Lysis
Cells are lysed in the prepared buffer under cold conditions to release intracellular proteins. Common lysis methods include sonication, liquid nitrogen grinding, or mechanical disruption. Throughout the procedure, samples must be kept at low temperatures to prevent enzymatic activity that could compromise phosphorylation.
3. Protein Collection
Following lysis, the samples are centrifuged to remove insoluble debris. The resulting supernatant contains soluble proteins, including phosphorylated forms, and is retained for downstream analysis.
4. Protein Quantification
Protein concentration is measured using standard assays such as BCA or Bradford to normalize sample input for subsequent experiments.
5. (Optional) Phosphoprotein Enrichment
To selectively enrich phosphorylated species, affinity-based techniques such as Immobilized Metal Affinity Chromatography (IMAC) or Titanium Dioxide (TiO₂) chromatography are employed. These methods exploit the specific binding affinities of phosphorylated residues for metal ions or metal oxides, allowing for targeted isolation from complex protein mixtures.
6. Storage
Extracted phosphoproteins should be stored at −80°C or under similarly low-temperature conditions to preserve phosphorylation and protein integrity. Proper storage ensures sample stability for future analyses.
Phosphorylated proteins obtained through this workflow can be further analyzed and validated using a combination of biochemical and proteomic techniques. These include SDS-PAGE for molecular weight and purity assessment, Western blotting with phosphorylation site-specific antibodies for expression profiling, and mass spectrometry for comprehensive mapping of phosphorylation sites and interaction networks.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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