2012 Promega catalogue

Cell Signaling
Streptavidin
Product Size Cat.# Price ($)
Streptavidin 1 mg Z7041 88.00
For Laboratory Use.
For additional information see page 10.
Streptavidin Alkaline Phosphatase
Product Size Cat.# Price ($)
Streptavidin Alkaline Phosphatase 0.5 ml V5591 250.00
For Laboratory Use.
For additional information see page 10.
Protein Interactions
CheckMate/Flexi ® Vector Mammalian Two-
Hybrid System
Product Size Cat.# Price ($)
CheckMate/Flexi ® Vector Mammalian Two-Hybrid
System 1 each C9360 975.00
Available Separately
pFN10A (ACT) Flexi ® Vector 20 µg C9331 489.00
pFN11A (BIND) Flexi ® Vector 20 µg C9341 489.00
pGL4.31[luc2P/GAL4UAS/Hygro] Vector 20 µg C9351 567.00
CheckMate Positive Control Vectors 1 set C9370 335.00
CheckMate Negative Control Vectors 1 set C9380 224.00
For Research Use Only. Not for Use in Diagnostic Procedures.
Description: The CheckMate/Flexi ® Vector Mammalian Two-Hybrid
System provides a means to confirm, validate and study suspected interactions
between two proteins or domains and can also be used to generate stable
cell lines for cell-based assays. Developed primarily for mammalian proteins
of interest, the system can allow protein expression and post-translational
modifications in an environment mimicking the native cell milieu. It is patterned
on the yeast two-hybrid system with one protein of interest (“X”) fused to a
DNA-binding domain and the other protein (“Y”) fused to a transcriptional
activation domain.
The system relies upon three plasmids that are co-transfected into
mammalian cells, each plasmid having unique features. The pFN10A (ACT)
Flexi ® Vector contains a herpes simplex virus VP16 transcriptional activation
domain upstream of the cloning site, and the pFN11A (BIND) Flexi ® Vector
contains the yeast GAL4DNA-binding domain upstream of the cloning site. The
pFN11A (BIND) Flexi ® Vector also expresses the Renilla reniformis luciferase
under the control of the SV40 promoter, allowing normalization for differences
in transfection efficiency. The third vector, pGL4.31[luc2P/GAL4UAS/Hygro]
Vector, contains five GAL4 binding sites upstream of a minimal TATA box, which
is upstream of a firefly luciferase gene that acts as a reporter for interactions
between proteins X and Y.
This system differs from the original CheckMate Mammalian Two-Hybrid
System in that the vectors are compatible with the Flexi ® Vector System, which
allows directional cloning and rapid, efficient and high-fidelity transfer of protein
coding regions between a variety of Flexi ® Vectors.
For complete and up-to-date product information visit: www.promega.com/catalog
Features:
• Mammalian-Based System: Interactions can be studied in the cell
line of choice. Proteins are more likely to be in their native conformation.
Post-translational modifications, such as glycosylation, phosphorylation and
acylation, are better maintained.
• Versatile: Vectors are based on the Flexi ® Cloning technology, enabling
convenient transfer of protein-coding regions for additional functional
proteomics applications.
• Convenient: The Dual-Luciferase ® Reporter Assay System is used for
detection.
Storage Conditions: Store at –20°C.
Protocol Part#
CheckMate/Flexi ® Vector Mammalian Two-Hybrid System
Technical Manual
TM283
CheckMate Mammalian Two-Hybrid
System
Product Size Cat.# Price ($)
CheckMate Mammalian Two-Hybrid System 1 system E2440 998.00
For Research Use Only. Not for Use in Diagnostic Procedures.
Description: Two-hybrid systems are extremely powerful methods for
detecting protein:protein interactions in vivo. The basis of two-hybrid systems
is the modular domains found in some transcription factors: a DNA-binding
domain, which binds to a specific DNA sequence, and a transcriptional
activation domain, which interacts with the basal transcriptional machinery. A
transcriptional activation domain in association with a DNA-binding domain will
promote the assembly of RNA polymerase II complexes at the TATA box and
increase transcription. In the CheckMate Mammalian Two-Hybrid System
the DNA-binding domain and the transcriptional activation domain, produced
by separate plasmids, are closely associated when one protein (“X”) fused
to a DNA-binding domain interacts with a second protein (“Y”) fused to a
transcriptional activation domain. In this system, interaction between proteins X
and Y results in transcription of a reporter gene.
Features:
• Mammalian System: Interactions can be studied in the cell line of choice.
Proteins are more likely to be in their native conformation. Post-translational
modifications, such as glycosylation, phosphorylation and acylation, are
better maintained.
• Convenient Quantitation: The Dual-Luciferase ® Reporter Assay System
is used for detection.
• Internal Control: Renilla luciferase normalizes transfection efficiency.
• Fast Transient Assay: Results obtained two days after transfection, as
compared to 3–4 days with the yeast system.
• Stable Transfectants: The pACT Vector contains the neomycin
phosphotransferase gene, which allows for selection of stable transfectants.
Storage Conditions: Store at –20°C.
Protocol Part#
CheckMate Mammalian Two-Hybrid System Technical Manual TM049
253
14
Protein Purification and Interactions
Section
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