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Chromatin Immunoprecipitation ChIP Workshop

Applications of ChIP, ChIP-Chip, and ChIP-Seq. Target Gene Identification (individual and global)Binding Site Motif IdentificationRNA--DNA--Protein InteractionsAnalysis of Epigenetic Phenomenon (chromatin structure, methylation). Starting ChIP. Treat cells with formaldehyde: protein-protein and p

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Chromatin Immunoprecipitation ChIP Workshop

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    1. Chromatin Immunoprecipitation (ChIP) Workshop Charlie Nicolet Heather N. Witt

    3. Starting ChIP Treat cells with formaldehyde: protein-protein and protein-DNA cross-links stop transcription factors in their tracks (DAY 0)

    6. Sonication using the BioRuptor!

    7. Sonication Check

    8. Immunoprecipitation (DAY 1 & 2) Y shaped molecules with binding sites for specific antigens. The heavy chain comes in several different classes. Hundreds of Abs to transcription factors are commercially available

    9. Five Classes of Antibodies

    10. Antibody Validation

    11. Positive and Negative Abs Positive 1º Ab = anti- wheat germ RNA PolII; murine MAb (DAY 1) 2º Ab = Rabbit Anti-Mouse IgG (DAY 2)

    12. Workshop Antibodies and DNA

    13. Staphylococcus aureus binding (DAY 2)

    14. Elution and Crosslink Reversal

    20. Acknowledgements Farnham lab Protocol Development Shally Xu Sharon Squazzo Cells & Materials Alina Rabinovich Shally Xu WGA and TCF4 ChIP-Seq Data Henny O’Geen

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