Electro Mobility Shift Assay

EMSA

Band Shift Assay

Gel Shift Assay

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Regulatory Region Coding Sequence

Gene Regulation by Transcription Factors

Application:

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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)

EMSA: Principle

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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.

NF-B

Free Probe

Radioaktively labeled oligonucleotidewith NF-B - binding site (probe) and bound NF-B

Radioactively labeled oligonucleotide with NF-B - binding site (probe)

Nuclear extract of non-activated cells

Nuclear extract ofactivated cells

Preparation of Nuclear and Cytosolic Extracts

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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 membrane3. Centrifuge to pellet nuclei4. Carefully remove supernatant (contains cytosolic and membrane fraction)4. Wash nuclear pellet once in lysis buffer5. Add hypertonic extraction buffer to nuclear pellet6. Agitate vigouresly for 30 minutes7. Centrifuge at high speed8. Remove nuclear extract, determine protein concentration and freeze on dry ice until EMSA is performed

The Probe

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Double stranded radiolabeled oligonucleotides containing a transcription factor binding site

AP-15’-GCT TGA TGA CTC AGC CGG AA C-3’3’-CGA ACT ACT GAG TCG GCC TT G-5’

NF-B5’-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.

Labeling the Probe (I)

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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 (-ATP)

Labeling the Probe (II)

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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

+ -32-P dGTP + dCTP + dTTP

Removal of Unincorporated Nucleotides

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Remove not incorporated nucleotides by Sephadex G50 column

or

non-denaturing PA gel purification

or

repeated ethanol precipitation

Reagents

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Competitor DNA: Competition of unspecificpoly (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

Analysis by non-DenaturingPolyacrylamide Gel

Electrophoresis

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Proof of Specificity

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Supershift using antibodies against the DNA-binding protein

Competition for binding to the radiolabeled probe usingunlabeled wildtype and mutated oligos

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Supershift

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.

p50/p65

Free probe

Radioaktiv labelled oligonucleotidewith NF-B - binding site (probe) and bound NF-B

Radioactiv labelled oligonucleotide with NF-B - binding site (probe)

Nuclear extract of activated cells

Nuclear extract of activated cells with anti-p50 antibody

p50/p65 + anti-p50

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Competition with Unlabeled Oligos

Increasing amounts of unlabeled oligos containing the NF-B binding siteor unlabeled oligos with a mutated binding site were added to the reaction mixprior to gel electrophoresis. Specific binding is extinguished only by the non-mutated oligo.

p50/p50

Free probe

p50/p65

Unspecific

Wild type oligo Mutated oligo

GGG GAC TTT CCC GGA GAC TTT CCC