Immunoprecipitation

Immunoprecipitation

1. Definition = Procedure which utilizes the specificity of an antibody for a specific protein in order to collect its interacting partners.

2. Types of Interactions

   • 1. Protein partners
   • 2. Ligands
   • 3. DNA sequences

3. Process

   • 1. Antibody is added to target protein
   • 2. Binding of identification protein
   • 4. Removal of supernatant
   • 3. Centrifuged
   • 5. Elute the sample (removal of antibodies)
   • 6. Run samples on electro gel
   • 7. IP (Immunoprecipitation) antibody added to one component of the protein and if other components appear on the gel ladder then they are interacting partners

4. Chromatin immunoprecipitation

   • Purpose
     • Identify DNA sequences which are bound by proteins of interest (transcriptional factors)
     • Chromatin IP may be used to determine the genomic geography of transcription factor binding.
   • Process
     • 1. Cross link proteins to DNA
     • 2. Shear DNA
     • 3. Bind to antibody and identification protein
     • 4. Purification of DNA
     • 5. Analyse DNA via = Microarray, Standard PCR, Sequencing

5. Constraints

   • Abundance of target
   • Antibody specificity
   • Sufficiently good binding to identification protein

6. IP may be further used to understand pathway dynamics