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Electro Mobility Shift Assay EMSA Band Shift Assay Gel Shift Assay PowerPoint Presentation
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Electro Mobility Shift Assay EMSA Band Shift Assay Gel Shift Assay

Electro Mobility Shift Assay EMSA Band Shift Assay Gel Shift Assay

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Electro Mobility Shift Assay EMSA Band Shift Assay Gel Shift Assay

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  1. Electro Mobility Shift AssayEMSABand Shift AssayGel Shift Assay R. Voll 09/01

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

  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) R. Voll 09/01

  4. Nuclear extract of activated cells Nuclear extract of non-activated cells EMSA: Principle 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

  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 R. Voll 09/01

  6. The Probe Double stranded radiolabeled oligonucleotides containing a transcription factor binding site AP-1 5’-GCT TGATGA CTC AGCCGG AA C-3’ 3’-CGA ACTACT GAG TCGGCC TT G-5’ NF-kB 5’-AGT TGAGGG GAC TTT CCCAGG C-3’ 3’-TCA ACTCCC CTC AAA GGGTCC G-5’ Binding motif R. Voll 09/01

  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. R. Voll 09/01

  8. Labeling the Probe (I) A. T4 Polynucleotide Kinase 5’-AGT TGAGGG GAC TTT CCCAGG-3’ 3’-CA ACTCCC CTC AAA GGGTCC G-5’ 5’-P-AGTTGA GGG GAC TTT CCCAGG-3’ 3’-CA ACTCCC CTC AAA GGGTCC G-P-5’ + Adenosin-P-P-P(g-ATP) PNK R. Voll 09/01

  9. Labeling the Probe (II) B. Klenow Fragment of E. coli DNA Polymerase I 5’-ACT TGAGGG GAC TTT CCCAG-3’ 3’-A ACTCCC CTC AAA GGGTCC G-5’ 5’-ACT TGAGGG GAC TTT CCCAGGC-3’ 3’-TGA ACTCCC CTC AAA GGGTCC G-5’ +a-32-P dGTP + dCTP + dTTP Klenow R. Voll 09/01

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

  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 R. Voll 09/01

  12. Analysis by non-DenaturingPolyacrylamide Gel Electrophoresis R. Voll 09/01

  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 R. Voll 09/01

  14. 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. R. Voll 09/01

  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. R. Voll 09/01