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Mammalian two-hybrid system

The Matchmaker Mammalian Assay Kit 2 allows rapid and convenient analysis of protein-protein interactions in transfected mammalian cells. The kit uses a SEAP secreted reporter to measure the interactions between the two proteins of interest, eliminating the need for cell lysis.

Because these two-hybrid assays are performed in mammalian cells, proteins encoded by mammalian cDNAs are more likely to be in their native conformation; therefore, post-translational modifications and experimental results are more likely to represent biologically significant interactions.

The Matchmaker Mammalian Two-Hybrid Assay Kit 2 allows rapid and convenient analysis of protein-protein interactions in transfected mammalian cells. The kit uses a SEAP secreted reporter to measure the interactions between the two proteins of interest, eliminating the need for cell lysis.

Because these two-hybrid assays are performed in mammalian cells, proteins encoded by mammalian cDNAs are more likely to be in their native conformation; therefore, post-translational modifications and experimental results are more likely to represent biologically significant interactions.

The Matchmaker Mammalian Assay Kit 2 is based on the same principle as the Matchmaker Gold Two-Hybrid System—two fusion proteins are expressed that contain either a DNA binding domain or an activation domain. If the proteins interact, transcription of the SEAP reporter gene is activated from the pG5-SEAP plasmid.

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Cat. # Product Size License Quantity Details
630305 Matchmaker® Mammalian Assay Kit 2 Each USD $846.00

Complete kit for confirming a known protein-protein interaction using two-hybrid technology in mammalian cell culture. Protein-protein interaction is assayed by measuring SEAP gene expression. No lysis is required.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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The mammalian two-hybrid principle

The mammalian two-hybrid principle
The mammalian two-hybrid principle. The bait protein is fused to the DNA binding domain from GAL4 and the prey protein is fused the transcriptional activation domain of VP16. If the two proteins interact at the PGAL4-E1b promoter (GAL promoter), SEAP is secreted into the growth medium.

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Chemiluminescent detection of protein-protein interactions

Chemiluminescent detection of protein-protein interactions
Chemiluminescent detection of protein-protein interactions. The bait vector pM-53 expresses p53 fused to the GAL4 DNA-binding domain. When HEK 293 cells were cotransfected with this bait together with the SV40 large T antigen prey vector, pVP16-T, strong expression of SEAP was detected because large T antigen interacts with p53. The CP protein, which does not interact strongly with p53, was used as a negative control.

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The Matchmaker Two-Hybrid Assay Kit 2 procedure

The Matchmaker Two-Hybrid Assay Kit 2 procedure
The Matchmaker Two-Hybrid Assay Kit 2 procedure.

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630305: Matchmaker Mammalian Assay Kit 2

630305: Matchmaker Mammalian Assay Kit 2

Required Products

Cat. # Product Size Price License Quantity Details
631736 Great EscAPe™ SEAP Chemiluminescence Kit 2.0 50 Rxns USD $280.00

The Great EscAPe SEAP Chemiluminescence Kit 2.0 is a complete system for the detection of secreted alkaline phosphatase (SEAP) activity in the growth media from mammalian cells expressing this enzyme. The kit contains reagents necessary for the chemiluminescent detection of SEAP activity using a 96-well plate luminometer. The assay can also be performed in single tubes and measured in a tube luminometer. It allows you to monitor promoter activity over time, because samples of the cell media can be taken repeatedly and tested without lysing cells.

Documents Components Image Data

Back

Chemiluminescent detection of protein-protein interactions

Chemiluminescent detection of protein-protein interactions
Chemiluminescent detection of protein-protein interactions. The bait vector pM-53 expresses p53 fused to the GAL4 DNA-binding domain. When HEK 293 cells were cotransfected with this bait together with the SV40 large T antigen prey vector, pVP16-T, strong expression of SEAP was detected because large T antigen interacts with p53. The CP protein, which does not interact strongly with p53, was used as a negative control.

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SEAP activity is highly specific, with a >20-fold increase in activity

SEAP activity is highly specific, with a >20-fold increase in activity

SEAP activity is highly specific, with a >20-fold increase in activity. HEK 293 cells were transiently transfected with a promoter construct containing CRE driving the expression of SEAP, or mock-transfected. 12 hr posttransfection, the cell culture media was replaced by media containing either 10 μM forskolin or plain cell culture media and incubated for 7 hr. Forskolin causes an increase in the level of cytosolic cAMP, which in turn activates CRE, driving the expression of SEAP, which is detectable in the media culture supernatant. The samples were assayed using the Great EscAPe SEAP Chemiluminescence Kit 2.0 and its protocol and analyzed on a BD Monolight 3096 Luminometer.

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Comparison of the sensitivities of SEAP and firefly luciferase

Comparison of the sensitivities of SEAP and firefly luciferase

Comparison of the sensitivities of SEAP and firefly luciferase. Parallel cultures of BHK cells were transiently transfected with the indicated amounts of either pSEAP2-Control Vector or a similar vector expressing luciferase. After 24 hr, SEAP activity was assayed in the appropriate culture media using the Great EscAPe chemiluminescent assay. Similarly, after 24 hr, cell lysates were prepared from the luciferase cultures, and luciferase activity was assayed using a commercial kit. RLU = relative light units.

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Signal transduction kits are available for in vivo studies of signal transduction—from signaling cascades to gene transcription

Signal transduction kits are available for in vivo studies of signal transduction—from signaling cascades to gene transcription
Signal transduction kits are available for in vivo studies of signal transduction—from signaling cascades to gene transcription.

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631736: Great EscAPe SEAP Chemiluminescence Kit 2.0

631736: Great EscAPe SEAP Chemiluminescence Kit 2.0

Overview

  • Fast, convenient analysis of protein-protein interactions in mammalian cells
  • SEAP secreted reporter eliminates the need for cell lysis
  • Biologically relevant results are more likely in a mammalian system

More Information

Applications

  • Confirm protein-protein interactions in mammalian cells
  • Map interacting domains

Additional product information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.

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