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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
Cells & Materials