Chromatin Immunoprecipitation ChIP Workshop

Chromatin Immunoprecipitation ChIP Workshop PowerPoint PPT Presentation


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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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