染色质免疫沉淀.ppt

Chromatin Immunoprecipitation(ChIP) Workshop
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
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!
Increases reproducibility between samples
Decreases contamination
Easy
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)
Antibodies are Y shaped molecules with binding sites for specific antigens.
The heavy chain e in several different classes.
Hundreds of Abs to transcription factors mercially available
Five Classes of Antibodies
IgA about 15% of total
antibody count.
IgM makes up 10% of our
total antibodies.
IgD less than 1%
IgE less than 1%
IgG approx. 75% of our serum
Ig pool, most polyclonals and
mAbs.
Antibody Validation
Step 1: Show reactivity on a Western (denatured epitope).
Step 2: Show IP ability. Perform IP, run Western and re-probe with same Ab (IP-West