DULIP: A Dual Luminescence-Based Co-Immunoprecipitation Assay for Interactome Mapping in Mammalian Cells.
Mapping of protein-protein interactions (PPIs) is critical for understanding protein function and complex biological processes. Here, we present DULIP, a dual luminescence-based co-immunoprecipitation assay, for systematic PPI mapping in mammalian cells. DULIP is a second-generation luminescence-based PPI screening method for the systematic and quantitative analysis of co-immunoprecipitations using two different luciferase tags. Benchmarking studies with positive and negative PPI reference sets revealed that DULIP allows the detection of interactions with high sensitivity and specificity. Furthermore, the analysis of a PPI reference set with known binding affinities demonstrated that both low- and high-affinity interactions can be detected with DULIP assays. Finally, using the well-characterized interaction between Syntaxin-1 and Munc18, we found that DULIP is capable of detecting the effects of point mutations on interaction strength. Taken together, our studies demonstrate that DULIP is a sensitive and reliable method of great utility for systematic interactome research. It can be applied for interaction screening and validation of PPIs in mammalian cells. Moreover, DULIP permits the specific analysis of mutation-dependent binding patterns.
Neuroproteomics, Max Delbrueck Center for Molecular Medicine, Robert-Roessle-Straße 10, 13125 Berlin, Germany.
Philipp
P
Trepte
Neuroproteomics, Max Delbrueck Center for Molecular Medicine, Robert-Roessle-Straße 10, 13125 Berlin, Germany.
Alexander
A
Buntru
Neuroproteomics, Max Delbrueck Center for Molecular Medicine, Robert-Roessle-Straße 10, 13125 Berlin, Germany.
Konrad
K
Klockmeier
Neuroproteomics, Max Delbrueck Center for Molecular Medicine, Robert-Roessle-Straße 10, 13125 Berlin, Germany.
Lindsay
L
Willmore
Neuroproteomics, Max Delbrueck Center for Molecular Medicine, Robert-Roessle-Straße 10, 13125 Berlin, Germany.
Anup
A
Arumughan
Neuroproteomics, Max Delbrueck Center for Molecular Medicine, Robert-Roessle-Straße 10, 13125 Berlin, Germany.
Christopher
C
Secker
Neuroproteomics, Max Delbrueck Center for Molecular Medicine, Robert-Roessle-Straße 10, 13125 Berlin, Germany.
Martina
M
Zenkner
Neuroproteomics, Max Delbrueck Center for Molecular Medicine, Robert-Roessle-Straße 10, 13125 Berlin, Germany.
Lydia
L
Brusendorf
Neuroproteomics, Max Delbrueck Center for Molecular Medicine, Robert-Roessle-Straße 10, 13125 Berlin, Germany.
Kirstin
K
Rau
Neuroproteomics, Max Delbrueck Center for Molecular Medicine, Robert-Roessle-Straße 10, 13125 Berlin, Germany.
Alexandra
A
Redel
Neuroproteomics, Max Delbrueck Center for Molecular Medicine, Robert-Roessle-Straße 10, 13125 Berlin, Germany. Electronic address: ewanker@mdc-berlin.de.
Erich E
EE
Wanker
D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods*; D008958:Models, Molecular; D051938:Munc18 Proteins / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D025941:Protein Interaction Mapping / Q000379:methods*; D050827:Syntaxin 1 / Q000235:genetics / Q000378:metabolism; D051025:bcl-Associated Death Protein / Q000378:metabolism; D051020:bcl-X Protein / Q000378:metabolism
D051938:Munc18 Proteins; D050827:Syntaxin 1; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein
detection of low- and high-affinity interactions; disease-mutation detection; luminescence normalization; quantitative interaction score and quantification of interaction strength; systematic protein–protein interaction screening