A microtiter assay for quantifying protein-protein interactions associated with cell-cell adhesion.
Cell-cell adhesions are a hallmark of epithelial tissues, and the disruption of these contacts plays a critical role in both the early and late stages of oncogenesis. The interaction between the transmembrane protein E-cadherin and the intracellular protein beta-catenin plays a crucial role in the formation and maintenance of epithelial cell-cell contacts and is known to be downregulated in many cancers. The authors have developed a protein complex enzyme-linked immunosorbent assay (ELISA) that can quantify the amount of beta-catenin bound to E-cadherin in unpurified whole-cell lysates with a Z' factor of 0.74. The quantitative nature of the E-cadherin:beta-catenin ELISA represents a dramatic improvement over the low-throughput assays currently used to characterize endogenous E-cadherin:beta-catenin complexes. In addition, the protein complex ELISA format is compatible with standard sandwich ELISAs for parallel measurements of total levels of endogenous E-cadherin and beta-catenin. In 2 case studies closely related to cancer cell biology, the authors use the protein complex ELISA and traditional sandwich ELISAs to provide a detailed, quantitative picture of the molecular changes occurring within adherens junctions in vivo. Because the E-cadherin: beta-catenin protein complex plays a crucial role in oncogenesis, this protein complex ELISA may prove to be a valuable quantitative prognostic marker of tumor progression.
Graham
Nicholas A
NA
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA.
Pope
Melissa D
MD
Rimchala
Tharathorn
T
Huang
Beijing K
BK
Asthagiri
Anand R
AR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D001943:Breast Neoplasms / Q000473:pathology; D015820:Cadherins / Q000032:analysis* / Q000378:metabolism / Q000502:physiology; D002448:Cell Adhesion; D002460:Cell Line; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000502:physiology*; D005260:Female; D006801:Humans; D007668:Kidney / Q000166:cytology; D051379:Mice; D010957:Plasmids; D011506:Proteins / Q000378:metabolism*; D015203:Reproducibility of Results; D012190:Retroviridae / Q000235:genetics; D014162:Transfection; D051176:beta Catenin / Q000032:analysis* / Q000378:metabolism / Q000502:physiology
D000911:Antibodies, Monoclonal; D015820:Cadherins; D011506:Proteins; D051176:beta Catenin