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Protein Interactions

Technical Resources

Pull-Down System
Footprinting
Gel Shifts & Nuclear Extracts
Primer Extension
Mammalian Two-Hybrid

In Vitro Transcription 
Protein Expression & Analysis
Protocols
Technical Articles
Pull-Down Assay Animation

OVERVIEW
Cell-free expression systems are popular tools for in vitro production of proteins in order to examine for their activity in protein:protein and protein:nucleic acid interactions. Often researchers will use the in vitro approach to verify in vivo results, for example, from a yeast two-hybrid screen. Putative DNA binding proteins synthesized in vitro can be used in an electrophoretic mobility shift assay (EMSA), where protein:nucleic acid complexes demonstrate retarded migration compared to their non-nucleic acid-binding counterparts. For a more in-depth discussion of in vitro protein synthesis and associated applications, please consult our In Vitro Expression Guide.

 

PULL-DOWN SYSTEM

MagneGST™ Pull-Down System

The MagneGST™ Pull-Down System (Cat.# V8870) provides GSH-linked magnetic particles that allow simple immobilization of GST-fusion bait proteins from bacterial lysates and an in vitro transcription/translation system for expression of prey proteins. Prey proteins are isolated using MagneGST™ (GSH) Particles containing the appropriate bait protein. The MagneGST™ Pull-Down System protocol can be used for detection of interactions between GST-fusion proteins expressed in bacterial lysates and prey proteins expressed in the TNT® System

Protocol:
MagneGST™ Pull-Down System (#TM249)

Technical Article:
Detection of Protein:Protein Interactions Using
   the MagneGST™ Pull-Down System
(PN087)



FOOTPRINTING

Core Footprinting System

The Core Footprinting System (Cat.# E3730) provides the reagents necessary to identify transcription factor binding sites, including a control transcription factor extract (AP2 Extract) and a purified SV40 Positive Control DNA in addition to the buffers and enzymes required to perform 5´ end-labeling and digestion of the DNA.

Protocol:
Core Footprinting System (#TB137)

Technical Articles:
Articles Citing Promega Products in Novel and Interesting Applications (PN067)
TFIID: What's in a name? (PN034)



GEL SHIFTS & NUCLEAR EXTRACTS

Gel Shift Assay System

The Gel Shift Assay System (Cat.# E3050, E3300) contains an AP2 Consensus Oligo, a SP1 Consensus Oligo, Gel Shift Binding 5X Buffer and sufficient HeLa Nuclear Extract to perform 20 control reactions. The Core System is a reliable system for obtaining experience with gel shift assays because AP2 binding activity is stable and produces a strong gel shift. The standard Gel Shift Assay System includes five additional oligonucleotides plus the components of the Core System.

Protocol:
Gel Shift Assay System (#TB110)

Technical Articles:
Gel Shift Assay System (PN045)
Non-Radioactive Gel Shift Assay (eNotes, AP0042)
pGEM®-T Easy Vector System is an Easy Tool for Preparing
   Gel Shift Probes
(eNotes, AP0039)


HeLaScribe® Nuclear Extract In Vitro Transcription System

The HeLaScribe® Nuclear Extract in vitro Transcription System (Cat.# E3521) includes all of the necessary components for eukaryotic in vitro transcription as well as a positive control template (CMV immediate early promoter DNA). HeLa nuclear extracts can support accurate transcription initiation by RNA polymerase II and exhibit both basal and regulated patterns of RNA polymerase transcription.

Protocol:
HeLaScribe® Nuclear Extract in vitro Transcription System (#TB123)

Technical Articles:
In Vitro Transcription on DNA Templates Immobilized to Streptavidin
    MagneSphere® Paramagnetic Particles
(PN064)
DNA Excision Repair Catalyzed in vitro by the HeLaScribe® Nuclear 
   Extract
(PN055)
Developing Meaningful Assays for the Eukaryotic Transcription Reagents 
   TFIIB and Anti-TFIIB
(PN045)


Transcription Factors and Consensus Oligonucleotides

Transcription Factors:
Recombinant Human AP1 (c-Jun) (Cat.# E3061) is the protein encoded by the human proto-oncogene, c-jun. Its expression is greatly modulated by extracellular stimuli such as phorbol ester and growth factor treatment. It is a member of the AP1 family. c-Jun is expressed in E. coli from a human cDNA clone and has a molecular weight of 40kDa.

Recombinant Human NF-κB (p50) (Cat.# E3770) is expressed in bacteria from a human cDNA derived from the full-length p105. p50, a processed product of its p105 precursor, is the first DNA-binding subunit of the NF-κB transcription factor to be described. The amino-terminal half of p50 has strong homology to the proto-oncogene, c-rel, and its viral counterpart, v-rel, and the Drosophila dorsal gene as well. 

Recombinant Human SP1 (rhSP1) (Cat.# E6391) is expressed from a human baculovirus cDNA clone in Sf9 cells. The SP1 transcriptional control sequence is found in a large number of viral and cellular promoters, including the HIV long terminal repeat. The SP1 transcription factor contains contiguous zinc finger motifs that provide specific DNA binding activity. SP1 factor interacts with other coactivator proteins that can modulate transcriptional activity. 

Recombinant Human TFIIB (rhTFIIB) (Cat.# E3790) is a general transcription factor involved in formation of an in vitro active complex capable of specifically initiating RNA synthesis by RNA polymerase II. 

Transcription Factor Consensus Oligonucleotides:
SP1, AP1, AP2, TFIID, NF-κB, OCT1 and CREB

Technical Articles:
Gel shift analysis with Human Recombinant AP1 (c-jun): 
   The effect of poly d(I-C) on specific complex formation
(PN037)
Transcriptional activation assays of recombinant transcription factors (PN034)
Using SAM Biotin Capture Membranes to Study Protein:DNA Binding (eNotes, AP0043)


PRIMER EXTENSION

Primer Extension System–AMV Reverse Transcriptase

Primer Extension System–AMV Reverse Transcriptase (Cat.# E3030) can be used to quantitate specific mRNA transcripts and map the start sites of transcription.

Protocol:
Primer Extension System–AMV Reverse Transcriptase (#TB113)


MAMMALIAN TWO-HYBRID

CheckMate™ Mammalian Two-Hybrid System

The CheckMate™ Mammalian Two-Hybrid System (Cat.# E2440) determines if one protein (“X”) fused to a DNA-binding domain interacts with a second protein (“Y”) fused to a transcriptional activation domain. If an interaction between proteins X and Y occurs, it results in transcription of a reporter gene. Positive control vectors are included with the system.

The two fusion plasmids in the CheckMate™ Mammalian Two-Hybrid System are the pBIND Vector, containing the yeast GAL4 DNA-binding domain upstream of a multiple cloning region, and the pACT Vector, containing the herpes simplex virus VP16 activation domain upstream of a multiple cloning region. The pG5luc Vector contains five GAL4 binding sites upstream of a minimal TATA box, which in turn is upstream of the firefly luciferase gene. When the constructs are transfected into the same 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.

Protocol:
CheckMate™ Mammalian Two-Hybrid System (#TM049)

Technical Article:
The CheckMate™ Mammalian Two-Hybrid System (PN066)

Vector Maps:
pACT Vector
pBIND Vector
pG5luc Vector




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