Electro Mobility Shift Assay EMSA Band Shift Assay Gel Shift Assay

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Electro Mobility Shift AssayEMSABand Shift
AssayGel Shift Assay
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Gene Regulation by Transcription Factors
Coding Sequence
Regulatory Region
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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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EMSA Principle
Nuclear extract of activated cells
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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Preparation of Nuclear and Cytosolic Extracts
The procedure is carried out on ice rsp at 4C
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-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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Annealing the Oligos
Heat up an equimolar mixture of the 2 oligos to
95C 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 5-P-AGT TGA GGG GAC TTT CCC AGG-3
3-CA ACT CCC CTC AAA GGG TCC G-P-5
PNK
Adenosin-P-P-P (g-ATP)

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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 5-ACT TGA GGG GAC
TTT CCC AGG C-3 3-TGA ACT CCC CTC AAA GGG TCC
G-5
Klenow
a-32-P dGTP dCTP dTTP

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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 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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Analysis by non-DenaturingPolyacrylamide 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 with anti-p50
antibody
Nuclear extract of activated cells
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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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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