Electro Mobility Shift Assay EMSA Band Shift Assay Gel Shift Assay R. Voll 09/01

Gene Regulation by Transcription Factors Regulatory Region Coding Sequence R. Voll 09/01

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) R. Voll 09/01

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. R. Voll 09/01

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 R. Voll 09/01

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 R. Voll 09/01

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. R. Voll 09/01

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 R. Voll 09/01

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 R. Voll 09/01

Removal of Unincorporated Nucleotides Remove not incorporated nucleotides by Sephadex G50 column or non-denaturing PA gel purification repeated ethanol precipitation R. Voll 09/01

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 R. Voll 09/01

Analysis by non-Denaturing Polyacrylamide Gel Electrophoresis R. Voll 09/01

Proof of Specificity Supershift using antibodies against the DNA-binding protein Competition for binding to the radiolabeled probe using unlabeled wildtype and mutated oligos R. Voll 09/01

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. R. Voll 09/01

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. R. Voll 09/01