CheckMate™ Mammalian Two-Hybrid System: Application Update

Assay principle for CheckMate™ Mammalian Two-Hybrid System.
Assay principle.

In the CheckMate™ Mammalian Two-Hybrid System, the pBIND Vector contains the yeast GAL4 DNA-binding domain upstream of a multiple cloning region, and the pACT Vector contains the herpes simplex virus VP16 activation domain upstream of a multiple cloning region. The two genes encoding the two potentially interactive proteins of interest are cloned into pBIND and pACT Vectors to generate fusion proteins with the DNA-binding domain of GAL4 and the activation domain of VP16, respectively. The pG5luc Vector contains five GAL4 binding sites upstream of a minimal TATA box, which in turn, is upstream of the firefly luciferase gene (luc+). The pGAL4 and pVP16 fusion constructs are transfected along with pG5luc Vector into mammalian cells. Interaction between the two test proteins, as GAL4 and VP16 fusion constructs, results in an increase in firefly luciferase expression over the negative controls. Traditionally mammalian two hybrid analysis was used to confirm initial data obtained from yeast two hybrid experiments.

Due to enhanced bioinformatics information and the development of improved co-immunoprecipiation/pulldown procedures/technology, there is a growing trend to use only mammalian cells to characterize protein:protein interactions. The following references illustrate the use of the CheckMate™ system to complement  other techniques to characterize protein;protein interactions using only mammalian cells .

Bagchi, P. et al. (2013) Molecular Mechanism behind Rotavirus Nsp-1 Mediated PI3 Kinase Activation: Interaction between NSP1 and the p85Subunit of PI3 kinase. J. Vir. 87, 2358-62.

Greninger, A. et al. (2013) ACBD3 Interaction with TBC1 domain 22 protein is differentially affected by Enteroviral and Kobuviral 3A protein binding.  mBio 4, 00098-13.

Patki, M. et al. ( 2013) The ETS Domain Transcription Factor ELK1 Direct a critical component of growth signalling by Androgen Receptor in prostrate cancer cells. J.Biol. Chem. 288 11047-65

Konig, H-G. (2012) Fibroblast growth factor homologous factor 1 interact with NEMO to regulate NF-kappaB signaling in neurons J. Cell Sci. 125, 6058-70

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Gary Kobs

Gary Kobs

Gary earned his B.S. in Bacteriology, UW-Madison in 1982. From 1982–1986 he served as Research Tech at UW-Madison. From 1986 to the present Gary has been with Promega Corporation serving in many capacities including as the very first editor of Promega Notes. He was also Manager Tech Services and Training, Product Manager Restriction/Modifying Enzymes, Product Manager Protein Analysis, and Sr. Product Manager for Protein Analysis products. Gary has retired from Promega Corporation.

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