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Matchmaker mammalian two hybrid assay kit

Manufactured by Takara Bio
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The Matchmaker Mammalian Two-Hybrid Assay Kit is a tool for studying protein-protein interactions in mammalian cells. It allows for the detection and analysis of interactions between two proteins of interest expressed in a mammalian cell line.

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Lab products found in correlation

Cell culture media, by Corning (2 mentions) Seap great escape chemiluminescence assay kit 2, by Takara Bio (2 mentions) Fetal bovine serum (fbs), by Corning (2 mentions) Metafectene pro transfection reagent, by Biontex (1 mentions) Enhanced chemiluminescence reagent, by GE Healthcare (1 mentions) Great escape seap chemiluminescence assay kit, by Takara Bio (1 mentions) Annexin 5 fitc apoptosis detection kit, by Merck Group (1 mentions) 2000 m camera system, by Kodak (1 mentions) Cat elisa kit, by Roche (1 mentions)

7 protocols using matchmaker mammalian two hybrid assay kit

1

Apoptosis Assay Protocol for Survivin and Caspase-3

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The antibodies against survivin (2808) and cleaved caspase 3 (9661) were purchased from Cell Signaling Technology. The matchmaker mammalian two hybrid assay kit and the Great Escape SEAP Chemiluminescence assay kit 2.0 were purchased from Clontech. The Annexin V-FITC apoptosis kit and the β-actin antibody (A5316) were purchased from Millipore-Sigma. The Metafectene Pro transfection reagent and the enhanced chemiluminescence reagent were from Biontex and GE Healthcare, respectively. Cell culture media and fetal bovine serum (FBS) were from Corning and Applied Biosystems-Life Technologies, respectively. All other chemicals were purchased from MilliporeSigma or Fisher Scientific.
Peery R., Cui Q., Kyei-Baffour K., Josephraj S., Huang C., Dong Z., Dai M., Zhang J.T, & Liu J.Y. (2022). A novel survivin dimerization inhibitor without a labile hydrazone linker induces spontaneous apoptosis and synergizes with docetaxel in prostate cancer cells. Bioorganic & medicinal chemistry, 65, 116761.
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2

Survivin modulation in Du145 cells

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The two-hybrid assay was performed as previously described22 (link) using the ClonTech Matchmaker Mammalian Two Hybrid Assay kit.25 (link) The two plasmid constructs containing survivin fused to the GAL4-DNA-binding domain and to the VP16 activation domain have been previously engineered.22 (link) Briefly, these two plasmids (0.45 μg each/well) along with the pGSEAP reporter plasmid (0.09 μg/well) and firefly luciferase plasmid (30 ng/well) were co-transfected into 1 × 105 Du145 cells per well in a 12 well plate. At 48 h after transfection, media were changed with fresh ones containing DMSO, 7F, or 7F1 for 24 h followed by determination of SEAP activity using the Takara ClonTech SEAP Great Escape chemiluminescence assay kit 2.0. Cells were then lysed to determine luciferase activity for transfection efficiency control.
Peery R., Cui Q., Kyei-Baffour K., Josephraj S., Huang C., Dong Z., Dai M., Zhang J.T, & Liu J.Y. (2022). A novel survivin dimerization inhibitor without a labile hydrazone linker induces spontaneous apoptosis and synergizes with docetaxel in prostate cancer cells. Bioorganic & medicinal chemistry, 65, 116761.
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3

Mammalian Two-Hybrid Assay for Survivin

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The mammalian two hybrid assay was performed using the ClonTech Matchmaker Mammalian Two Hybrid Assay kit with the two newly engineered fusion plasmids as described above. Briefly, DU145 cells were seeded in 12-well plates at 1 × 105 cells/well and cultured for 24 h followed by transfection using pBIND-survivin and pACT-survivin (0.45 μg/well) along with the pGSEAP reporter plasmid (0.09 μg/well) and firefly luciferase reporter plasmid (30 ng/well). Two days after transfection, media was changed and cells were treated with DMSO, LQZ-7, or LQZ-7 analogues for 24 h followed by determination of SEAP activity using the Takara ClonTech SEAP Great Escape chemiluminescence assay kit 2.0. Luciferase activity was also measured as a control for transfection efficiency.
Peery R., Kyei-Baffour K., Dong Z., Liu J., de Andrade Horn P., Dai M., Liu J.Y, & Zhang J.T. (2020). Synthesis and Identification of a Novel Lead Targeting Survivin Dimerization for Proteasome-Dependent Degradation. Journal of medicinal chemistry, 63(13), 7243-7251.
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4

Molecular Biology Assay Reagents

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Antibodies against HA tag (H6908) and actin (A5316) and the Annexin V-FITC apoptosis detection kit were obtained from MilliporeSigma. The antibody against survivin (2808) and the Metafectene Pro transfection reagent were purchased from Cell Signaling Technology and Bion-tex, respectively. The enhanced chemiluminescence reagent was from GE Healthcare. The matchmaker mammalian two hybrid assay kit and the Great Escape SEAP Chemiluminescence assay kit were purchased from Clontech. Cell culture media and fetal bovine serum were from Corning and Applied Biosystems-Life Technologies, respectively. All other chemicals were purchased from Sigma or Fisher Scientific.
Peery R., Kyei-Baffour K., Dong Z., Liu J., de Andrade Horn P., Dai M., Liu J.Y, & Zhang J.T. (2020). Synthesis and Identification of a Novel Lead Targeting Survivin Dimerization for Proteasome-Dependent Degradation. Journal of medicinal chemistry, 63(13), 7243-7251.
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5

Mammalian Two-Hybrid Assay for NCC-ENaC Interaction

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We utilized the Matchmaker Mammalian Two-hybrid assay kit (Clontech). A full-length clone for mouse NCC was inserted into the DNA-binding domain (DNA-BD) plasmid vector, pM. Full-length clones of each subunit of ENaC were inserted into the activation domain (AD) plasmid, pVP16. These constructs were transfected into CHO cells along with the pG5CAT reporter plasmid. Each ENaC subunit was tested singly with NCC. The reporter gene will only be expressed if the protein encoded in the DNA-BD vector binds to the protein encoded in the AD vector. 48 hours after transfection cells were harvested and binding was assessed by measuring reporter gene expression (chloramphenicol acetyltransferase, CAT). Experiments were analyzed for the presence of chloramphenicol acetyltransferase (CAT) with a commercially available UV assay (FAST CAT Assay Kit, Thermo-Fisher, Molecular Probes, Cat # F-6616). Blots were visualized with a Kodak 2000M camera system (Eastman Kodak). Intensity of signal was detected with Image J.
Mistry A.C., Wynne B.M., Yu L., Tomilin V., Yue Q., Zhou Y., Al-Khalili O., Mallick R., Cai H., Alli A.A., Ko B., Mattheyses A., Bao H.F., Pochynyuk O., Theilig F., Eaton D.C, & Hoover R.S. (2016). The Sodium Chloride Cotransporter (NCC) and Epithelial Sodium Channel (ENaC) Associate. The Biochemical journal, 473(19), 3237-3252.
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6

Two-Hybrid Assay for p53-GLI1 Interaction

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The two-hybrid assay was performed using the mammalian matchmaker two-hybrid assay kit (Clontech, Mountain View, CA). HeLa cells were co-transfected with pM-p53 (Clontech, Mountain View, CA), pVP-16-GLI1, pG5CAT, and the pSV40β-GAL reporter. Cell lysates were prepared 48 h after transfection. β-galactosidase activity was used to normalize CAT activity.
Yoon J.W., Lamm M., Iannaccone S., Higashiyama N., Leong K.F., Iannaccone P, & Walterhouse D. (2015). P53 Modulates The Activity Of The GLI1 Oncogene Through Interactions With The Shared Coactivator TAF9. DNA repair, 34, 9-17.
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7

Mammalian Matchmaker Two-Hybrid Assay

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The mammalian Matchmaker two-hybrid assay kit (Clontech) was used to generate the desired plasmid constructs. The human PEX5 gene was amplified by PCR with oligonucleotides RE3803 and RE3802 and pGD106 [54 (link)] as template. The resulting PCR-fragment was subcloned in the NotI/SacII- restricted pVP16 expression Vector, containing the VP16-activation domain. USP2 Isoform 3 and its variants were cloned in the pM vector, carrying the GAL4-binding domain. USP2-3 was amplified with the oligonucleotides RE3541 and RE3536 and subcloned into SmaI/HindIII digested pM. USP2-3 ΔSRM and USP2-3 SRM>SKL were amplified with a different reverse primers, RE3763 and RE4330, respectively and also cloned into pM via SmaI/HindIII.
HEK-293 cells were cultured and transfected as described above. For the mammalian two-hybrid assay, all three plasmids, pM-USP2-3 variant (0.9 μg), pVP16-PEX5 (0.9 μg), and pG5CAT reporter vector (0.2 μg) were co-transfected. The cells were allowed to grow at 37°C in 8% CO2 for 48 h. After transfection, secreted chloramphenicol transferase (CAT) was monitored using the CAT ELISA kit (Roche Diagnostics, Mannheim, Germany).
Reglinski K., Keil M., Altendorf S., Waithe D., Eggeling C., Schliebs W, & Erdmann R. (2015). Peroxisomal Import Reduces the Proapoptotic Activity of Deubiquitinating Enzyme USP2. PLoS ONE, 10(10), e0140685.
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