Protocols and Applications Guide (US Letter Size) - Promega

||||||||||| 11Protein Purification and Analysis
Additional Resources for the MagneGST Pull-Down
System
Technical Bulletins and Manuals
TM249 MagneGST Pull-Down System Technical
Manual
(www.promega.com
/tbs/tm249/tm249.html)
Promega Publications
PN087 Detection of protein:protein interactions
using the MagneGST Pull-Down System
(www.promega.com
/pnotes/87/11527_23/11527_23.html)
VII. Analysis of DNA:Protein Interactions
Regulation of chromatin structure and gene expression is
essential for normal development and cellular growth.
Transcriptional events are tightly controlled both spatially
and temporally by specific protein:DNA interactions.
Currently there is a rapidly growing trend toward
genome-wide identification of protein-binding sites on
chromatin to characterize regulatory protein:DNA
interactions that govern the transcriptome. Common
methods to examine protein:DNA interactions include the
electrophoretic mobility shift assay, also known as the gel
shift assay, and chromatin immunoprecipitation (Solomon
et al. 1985; Solomon et al. 1988) coupled with DNA
microarray or ultrahigh-throughput sequencing analysis.
A. Gel Shift Assays
Electrophoretic mobility shift assays (EMSA) or gel shift
assays can be used to analyze protein:DNA complexes
expressed in vitro. The proteins are incubated with an
oligonucleotide containing a target consensus sequence
site, and DNA binding is detected by gel shift. An animated
presentation (www.promega.com
/paguide/animation/selector.htm?coreName=tnt02)
(requires Flash plug in) of protein:DNA interaction
detection using the TNT® Systems and Gel Shift Assay is
available. The gel shift assay provides a simple and rapid
method to detect DNA-binding proteins (Ausubel et al.
1989). This method is used widely in the study of
sequence-specific DNA-binding proteins such as
transcription factors. The assay is based on the observation
that complexes of protein and DNA migrate through a
nondenaturing polyacrylamide gel more slowly than free
DNA fragments or double-stranded oligonucleotides. The
gel shift assay is performed by incubating a purified
protein, or a complex mixture of proteins (such as nuclear
or cell extract preparations), with a 32P end-labeled DNA
fragment containing the putative protein-binding site. The
reaction products are then analyzed on a nondenaturing
polyacrylamide gel. The specificity of the DNA-binding
protein for the putative binding site is established by
competition experiments using DNA fragments or
oligonucleotides containing a binding site for the protein
of interest or other unrelated DNA sequences.
Protocols & Applications Guide
www.promega.com
rev. 3/09
Promega gel shift assay systems contain target
oligonucleotides, a control extract containing DNA-binding
proteins, binding buffer and reagents for phosphorylating
oligonucleotides. The Gel Shift Assay Core System (Cat.#
E3050) includes sufficient HeLa nuclear extract to perform
20 control reactions, Gel Shift Binding 5X Buffer, an SP1
Consensus Oligo and an AP2 Consensus Oligo. The
complete Gel Shift Assay System (Cat.# E3300) contains
five additional double-stranded oligonucleotides that
represent consensus binding sites for AP1, NF-κB, OCT1,
CREB and TFIID. These oligonucleotides can be end-labeled
and used as protein-specific probes or as specific or
nonspecific competitor DNA in competition assays. A
detailed protocol is available in Technical Bulletin #TB110
(www.promega.com/tbs/tb110/tb110.html).
Additional Resources for Gel Shift Assay Systems
Technical Bulletins and Manuals
TB110 Gel Shift Assay Systems Technical Bulletin
(www.promega.com/tbs/tb110/tb110.html)
Promega Publications
PN037 Gel shift analysis with Human
Recombinant AP1 (c-jun): The effect of
poly d(I-C) on specific complex formation
(www.promega.com
/pnotes/37/37_14/37_14.htm)
PN045 Technically speaking: Gel Shift Assay
System
(www.promega.com
/pnotes/45/45p25/45p25.html)
Citations
Lee, J. et al. (2002) Kaurane diterpene, kamebakaurin,
inhibits NF-kappa B by directly targeting the DNA-binding
activity of p50 and blocks the expression of antiapoptotic
NF-kappa B target genes J. Biol. Chem. 277, 18411–20.
To investigate the effect of the compound kamebakaurin
(KA) on NF-κB, an NF-κB-responsive firefly luciferase
vector was transfected into HeLa, Jurkat and THP-1 cells.
The Luciferase Assay System was used to assay the level
of NF-κB induction after treatment of cells with various
concentrations of KA. To determine if KA influenced the
DNA-binding activity of NF-κB, nuclear extracts of HeLa,
Jurkat and THP-1 cells were prepared after preincubation
with KA and stimulation of NF-κB activity. Control nuclear
extracts were prepared from unstimulated p50- or
RelA-overexpressed MCF-7 cells. In addition, the wildtype
and DNA-binding mutant RelA and p50 (NF-κB)
His-tagged proteins were translated using the TNT® Quick
Coupled Transcription/Translation System and
subsequently purified. Using the Gel Shift Assay System,
the NF-κB and AP1 oligos were tested for electromobility
shifts with the prepared nuclear extracts or with purified
wildtype and mutant proteins. Supershift studies using
anti-p50 or anti-RelA antibodies were also performed
PubMed Number: 11877450
PROTOCOLS & APPLICATIONS GUIDE 11-19