Kinetics of protein-protein interactions of connexins: use of enzyme linked sorbent assays.
Determination of the protein-protein interactions of connexins has become a rapidly expanding field of research. While there are multiple methods of determining the identity of binding partners, determination of the strengths of interactions is not as simple. Here we describe the use of the in vitro method Enzyme Linked Sorbent Assay (ELSA) to compare binding affinities of known protein partners for Connexin43. We used the binding of Cx43 Carboxyl Terminal domain to the PDZ-2 domain of Zonula Occludens-1 and to the SH3 domain of c-Src. In the ELSA assay we found that while the binding of the SH3 domain of c-Src is pH-dependent, the interaction of the PDZ domain of ZO-1 is not. These data confirm findings using Surface Plasmon Resonance (1) and indicate that ELSA can be a useful tool in determining the kinetics of protein-protein interactions.
Duffy
Heather S
HS
Albert Einstein College of Medicine, Bronx, New York 10461, USA. hduffy@aecom.yu.edu
O'Donnell
Phyllis
P
Coombs
Wanda
W
Taffet
Steven M
SM
Delmar
Mario
M
Spray
David C
DC
D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D018031:Connexin 43 / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008968:Molecular Conformation; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats; D062826:Zonula Occludens-1 Protein; D018909:src Homology Domains / Q000502:physiology*
D018031:Connexin 43; D008565:Membrane Proteins; D010750:Phosphoproteins; C569042:TJP1 protein, human; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein