1. Electro Mobility Shift Assay EMSA Band Shift Assay Gel Shift Assay
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2. Gene Regulation by Transcription Factors
Regulatory Region
Coding Sequence
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3. 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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4. EMSA: Principle Nuclear extract of non-activated cells
Radioaktively labeled oligonucleotide
with NF-B - binding site (probe)
and bound NF-B
NF-B
Radioactively labeled oligonucleotide
with NF-B - binding site (probe)
Free 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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5. 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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6. 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-kB
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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7. 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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8. 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’
5’-P-AGT TGA GGG GAC TTT CCC AGG-3’
3’-CA ACT CCC CTC AAA GGG TCC G-P-5’
+ Adenosin-P-P-P (g-ATP)
PNK
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9. 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’
5’-ACT TGA GGG GAC TTT CCC AGG C-3’
3’-TGA ACT CCC CTC AAA GGG TCC G-5’
+ a-32-P dGTP + dCTP + dTTP
Klenow
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10. Removal of Unincorporated Nucleotides
Remove not incorporated nucleotides by
Sephadex G50 column or
non-denaturing PA gel purification
repeated ethanol precipitation
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11. Reagents Competitor DNA: Competition of unspecific
poly (dI-dC) . poly (dI-dC) 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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12. Analysis by non-Denaturing Polyacrylamide Gel Electrophoresis
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13. 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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14. Supershift Nuclear extract of activated cells
cells with anti-p50 antibody
Supershift
p50/p65 + anti-p50
Radioaktiv labelled oligonucleotide
with NF-B - binding site (probe)
and bound NF-B
p50/p65
Radioactiv labelled oligonucleotide
with NF-B - binding site (probe)
Free 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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15. Competition with Unlabeled Oligos
p50/p65
p50/p50
Unspecific
Free probe
GGG GAC TTT CCC
GGA GAC TTT CCC
Wild type oligo Mutated oligo
Increasing amounts of unlabeled oligos containing the NF-kB 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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