A miniaturized sandwich immunoassay platform for the detection of protein-protein interactions.
BACKGROUND
Analysis of protein-protein interactions (PPIs) is a valuable approach for the characterization of huge networks of protein complexes or proteins of unknown function. Co-immunoprecipitation (coIP) using affinity resins coupled to protein A/G is the most widely used method for PPI detection. However, this traditional large scale resin-based coIP is too laborious and time consuming. To overcome this problem, we developed a miniaturized sandwich immunoassay platform (MSIP) by combining antibody array technology and coIP methods.


RESULTS
Based on anti-FLAG antibody spotted aldehyde slides, MSIP enables simple, rapid and large scale detection of PPIs by fluorescent labeling anti-myc antibody. By analyzing well-known interacting and non-interacting protein pairs, MSIP was demonstrated to be highly accurate and reproducible. Compared to traditional resin-based coIP, MSIP results in higher sensitivity and enhanced throughput, with the additional benefit of digital read-outs. In addition, MSIP was shown to be a highly useful validation platform to confirm PPI candidates that have been identified from yeast two hybrid systems.


CONCLUSIONS
In conclusion, MSIP is proved to be a simple, cost-saving and highly efficient technique for the comprehensive study of PPIs.
Liu
Qiongming
Q
State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China.
Chen
Qing
Q
Wang
Jian
J
Zhang
Ying
Y
Zhou
Ying
Y
Lin
Cong
C
He
Wei
W
He
Fuchu
F
Xu
Danke
D
D057809:HEK293 Cells; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D047468:Immunoprecipitation / Q000379:methods; D040081:Protein Array Analysis / Q000379:methods; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D011506:Proteins