Protein interactions (and analysis)

Protein interactions (and analysis)

1. Measuring the strength of protein interactions

   • Theory
   • Typical applications
     • Can be used to measure any protein-protein interaction in solution or on a surface providing you have a suitable detection system
       • PAGE (Poly-acrylamide Gel Electrophoresis)
       • Western Blot
       • Far western or blot overlay
       • ELISA (Enzyme-Linked Immunosorbent Assay

2. Surface plasmon resonance

   • aka ‘Biacore’
   • The Kd of a given protein-protein interaction can be read directly from traces like these
     • however one limitation is the attachment of proteins to a solid support which may cause conformational change and affect the result

3. Genetic methods of identifying protein interactions

   • Phage display libraries
     • Use of random peptide libraries is a powerful technique with many applications
       • Identification of specific epitopes as drug or antibody targets, mapping of protein-protein binding interfaces, generation of artificial antibodies, epitope mapping of antibodies to identify important binding sites etc.
   • Yeast two-hybrid screening
     • Bait protein cDNA cloned into bait plasmid in the host yeast strain.
       • Prey usually a whole library of cDNA’s transfected with the bait plasmid.
         • Interaction between bait and prey allows DNA binding and activation domains to activate a reporter gene.
           • Yeast colony survives on a restrictive growth media.
             • Prey plasmid recovered and sequenced to find interacting protein.

4. Electron Microscopy II - actin

   • EM of actin filaments with a binding protein attached.
     • EM uses negative stain (shown) or vitreous ice (cryo-EM) to preserve the specimen.
       • Image analysis is then employed to build up an average structure.

5. A protein machine of the cell - actomyosin

   • Muscle ultrastructure

Images and files in the map