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Chromatin Immunoprecipitation ChIP Workshop

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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) Antibodies are Y shaped molecules with binding sites for specific antigens. The heavy chain can come 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 Data Henny O’Geen

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