Electro Mobility Shift Assay
EMSA
Band Shift Assay
Gel Shift Assay
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Gene Regulation by Transcription Factors
Regulatory Region
Coding Sequence
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Application:
Detection of DNA-binding factors/proteins
•Analysis of DNA sequences (e. g. promoter or
enhancer regions) for their potential to bind
specifically to proteins/nuclear extracts
•Analysis of (sub-)cellular extracts for the presence
of certain DNA-binding proteins (e. g. a transcription
factor with a known recognition sequence)
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Nuclear extract
of non-activated
cells
Nuclear extract of
activated cells
EMSA: Principle
NF-B
Radioaktively labeled oligonucleotide
with NF-B - binding site (probe)
and bound NF-B
Free Probe
Radioactively labeled oligonucleotide
with NF-B - binding site (probe)
A double-stranded oligonucleotide containig a NF-B- binding site is labeled
with a radioactive isotope and incubated with a nuclear extract.
During gel-electrophoresis, NF-B bound to the oligonucleotide causes a shift
compared to the free probe.
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Preparation of Nuclear and Cytosolic Extracts
The procedure is carried out on ice rsp at 4°C and in the
presence of protease (and phosphatase) inhibitors.
1. Swell cells in hypotonic lysis buffer
2. Add NP-40 and vortex to disrupt cytoplasmic membrane
3. Centrifuge to pellet nuclei
4. Carefully remove supernatant (contains cytosolic and
membrane fraction)
4. Wash nuclear pellet once in lysis buffer
5. Add hypertonic extraction buffer to nuclear pellet
6. Agitate vigouresly for 30 minutes
7. Centrifuge at high speed
8. Remove nuclear extract, determine protein concentration
and freeze on dry ice until EMSA is performed
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The Probe
Double stranded radiolabeled oligonucleotides
containing a transcription factor binding site
AP-1
5’-GCT TGA TGA CTC AGC CGG AA C-3’
3’-CGA ACT ACT GAG TCG GCC TT G-5’
NF-B
5’-AGT TGA GGG GAC TTT CCC AGG C-3’
3’-TCA ACT CCC CTC AAA GGG TCC G-5’
Binding motif
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Annealing the Oligos
Heat up an equimolar mixture of the 2 oligos
to 95°C and let them slowly cool down by turning
off the heat block.
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Labeling the Probe (I)
A. T4 Polynucleotide Kinase
5’-AGT TGA GGG GAC TTT CCC AGG-3’
3’-CA ACT CCC CTC AAA GGG TCC G-5’
+ Adenosin-P-P-P (g-ATP)
PNK
5’-P-AGT TGA GGG GAC TTT CCC AGG-3’
3’-CA ACT CCC CTC AAA GGG TCC G-P-5’
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Labeling the Probe (II)
B. Klenow Fragment of E. coli DNA Polymerase I
5’-ACT TGA GGG GAC TTT CCC AG-3’
3’-A ACT CCC CTC AAA GGG TCC G-5’
+ a-32-P dGTP + dCTP + dTTP
Klenow
5’-ACT TGA GGG GAC TTT CCC AGG C-3’
3’-TGA ACT CCC CTC AAA GGG TCC G-5’
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Removal of Unincorporated
Nucleotides
Remove not incorporated nucleotides by
Sephadex G50 column
or
non-denaturing PA gel purification
or
repeated ethanol precipitation
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Reagents
Competitor DNA:
poly (dI-dC) . poly (dI-dC)
Competition of unspecific
binding (e. g. histones)
BSA:
Protection of nuclear
extracts
GTP:
?
Radiolabeled Probe:
Detection of DNA-binding
proteins
Reaction Buffer
Binding conditions
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Analysis by non-Denaturing
Polyacrylamide Gel
Electrophoresis
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Proof of Specificity
Supershift using antibodies against the DNA-binding
protein
Competition for binding to the radiolabeled probe using
unlabeled wildtype and mutated oligos
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Nuclear extract
of activated cells
Nuclear extract of activated
cells with anti-p50 antibody
Supershift
p50/p65 + anti-p50
p50/p65
Free probe
Radioaktiv labelled oligonucleotide
with NF-B - binding site (probe)
and bound NF-B
Radioactiv labelled oligonucleotide
with NF-B - binding site (probe)
A double-stranded oligonucleotide containig a NF-B- binding site is labelled
radioactive and incubated with a nuclear extract.
During gel-electrophoresis, NF-B bound to the oligonucleotide causes a shift
compared to the free probe.
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Competition with Unlabeled Oligos
p50/p65
p50/p50
Unspecific
Free probe
GGG GAC TTT CCC
Wild type oligo
GGA GAC TTT CCC
Mutated oligo
Increasing amounts of unlabeled oligos containing the NF-B binding site
or unlabeled oligos with a mutated binding site were added to the reaction mix
prior to gel electrophoresis. Specific binding is extinguished only by the
non-mutated oligo.
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