染色质免疫沉淀.ppt

Chromatin Immunoprecipitation ( ChIP ) Workshop Chromatin Immunoprecipitation ( ChIP ) Workshop Charlie Nicolet Heather N. Witt Charlie Nicolet Heather N. Witt Applications of ChIP, ChIP-Chip, and ChIP-Seq ? Target Gene Identification (individual and global) ? Binding Site Consensus Identification ? RNA--DNA--Protein Interactions ? Analysis of ic Phenomenon (eg, methylation, silencing) Starting ChIP Starting ChIP ? Treat cells with formaldehyde: protein-protein and protein-DNA cross-links stop transcription factors in their tracks (DAY 0) ? Treat cells with formaldehyde: protein-protein and protein-DNA cross-links stop transcription factors in their tracks (DAY 0) 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 Sonication using the BioRuptor ! Sonication using the BioRuptor ! ? Increases reproducibility between samples ? Decreases contamination ? Easy Sonication Check Sonication Check 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 kbp Fragment size limits for Qiaquick PCR Purification Column is 100 bp - 10 kb. 100 bp Immunoprecipitation (DAY 1 & 2) 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