Immunoprecipitation of Phosphorylated Proteins

Immunoprecipitation (IP) is an experimental technique used for the isolation and purification of specific proteins and their interaction partners, often used in the study of phosphorylated proteins. By using an antibody to specifically bind to the target protein or phosphorylation site, specific proteins or protein complexes can be enriched from complex biological samples. When applied to the study of phosphorylated proteins, this technique is known as phosphorylated protein immunoprecipitation.

 

Analysis Workflow

1. Antibody Selection

Choose a highly specific antibody that can specifically recognize the target protein or its phosphorylation site.

 

2. Sample Preparation

Cell or tissue samples are lysed to extract proteins, and then cell debris is cleared through steps such as centrifugation.

 

3. Immunoprecipitation

The specific antibody is added to the protein sample, allowing the antibody to bind with the target protein. Then, substances with high affinity to the antibody Fc region, such as Protein A or Protein G, are added to precipitate the antibody-protein complex.

 

4. Wash and Analysis

Non-specifically bound proteins are removed by washing, and the precipitate is analyzed by Western Blot, Mass Spectrometry, or other methods.

 

Applications

1. Identification and Verification of Phosphorylated Proteins

Specific phosphorylated proteins can be enriched using anti-phospho specific antibodies (such as those against phosphorylated tyrosine, serine or threonine), for identification and functional studies.

 

2. Signal Pathway Analysis

By enriching proteins that are phosphorylated under specific cellular stimulation, key nodes and regulatory mechanisms in signal transduction pathways can be revealed.

 

3. Study of Dynamic Changes in Phosphorylation

By immunoprecipitating and subsequently analyzing phosphorylated proteins at different time points, dynamic changes in phosphorylation status can be studied, understanding how proteins respond to intra- and extracellular signals.