染色质免疫与沉淀.pptVIP

Chromatin Immunoprecipitation(ChIP) Workshop Charlie Nicolet Heather N. Witt Starting ChIP Treat cells with formaldehyde: protein-protein and protein-DNA cross-links stop transcription factors in their tracks (DAY 0) Sonication using the BioRuptor!  Sonication Check 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 Five Classes of Antibodies Antibody Validation Positive and Negative Abs Positive 1o Ab = anti- wheat germ RNA PolII; murine MAb (DAY 1) 2o Ab = Rabbit Anti-Mouse IgG (DAY 2) Workshop Antibodies and DNA Staphylococcus aureus binding (DAY 2) Elution and Crosslink Reversal Acknowledgements Farnham lab Protocol Development Shally Xu Sharon Squazzo Cells Materials Alina Rabinovich Shally Xu WGA Data Henny O’Geen * * Applications of ChIP, ChIP-Chip, and ChIP-Seq Target Gene Identification (individual and global) Binding Site Consensus Identification RNA--DNA--Protein Interactions Analysis of Epigenetic Phenomenon (eg, methylation, silencing) Cross-linking can be done on: Suspension cells Adherent Cells Tissues Anatomical structures See protocol and web page for additional information on cross-linking and example protocols Formaldehyde Crosslinking Protein-Protein DNA-DNA Other Options Douncing Dounce contains two sizes A is large clearance B is small clearance Force of moving rod B in dounce results: disperses cell clumps “pokes” holes in membrane breaks cell membrane to release nuclei ChIP--Day 1 Swelling Buffer is used to bloat cell to allow cell membrane to break more easily during Douncing Increases reproducibility between samples Decreases contamination Easy Sonication results in many different sized fragments and should be verified for your experiment 6 pulses 11 pulses 21 pulses 31 pulses 500 bp 10 kbp 3 kb