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1、染色染色质质免疫沉淀免疫沉淀第1页，共21页，编辑于2022年，星期六Applications of ChIP,ChIP-Chip,and ChIP-SeqTarget Gene Identification(individual and global)Binding Site Consensus IdentificationRNA-DNA-Protein InteractionsAnalysis of Epigenetic Phenomenon(eg,methylation,silencing)第2页，共21页，编辑于2022年，星期六Starting ChIPStarting ChIP T
2、reat cells with formaldehyde:protein-Treat cells with formaldehyde:protein-protein and protein-DNA cross-links protein and protein-DNA cross-links stop transcription factors in their tracks stop transcription factors in their tracks (DAY 0)(DAY 0)Cross-linking can be done on:Suspension cellsAdherent
3、 CellsTissuesAnatomical structuresSee protocol and web page for additional information on cross-linking and example protocols第3页，共21页，编辑于2022年，星期六Formaldehyde CrosslinkingProtein-ProteinDNA-DNAOther Options第4页，共21页，编辑于2022年，星期六 DouncingvDounce contains two sizes A is large clearance B is small clear
4、ancevForce of moving rod B in dounce results:disperses cell clumps“pokes”holes in membrane breaks cell membrane to release nucleiChIP-Day 1Swelling Buffer is used to bloat cell to allow cell membrane to break more easily during Douncing第5页，共21页，编辑于2022年，星期六Sonication using the BioRuptor!Sonication u
5、sing the BioRuptor!Increases reproducibility between samples Decreases contamination Easy第6页，共21页，编辑于2022年，星期六Sonication CheckSonication CheckSonication results in many different sized fragments and should be verified for your experiment 6 pulses11 pulses21 pulses31 pulses500 bp10 kbp3 kbpFragment s
6、ize limits for Qiaquick PCR Purification Column is 100 bp-10 kb.100 bp第7页，共21页，编辑于2022年，星期六Immunoprecipitation(DAY 1&2)Immunoprecipitation(DAY 1&2)Antibodies are Y shaped Antibodies are Y shaped molecules with binding molecules with binding sites for specific antigens.sites for specific antigens.The
7、 heavy chain can The heavy chain can come in several different come in several different classes.classes.Hundreds of Abs to Hundreds of Abs to transcription factors are transcription factors are commercially availablecommercially available第8页，共21页，编辑于2022年，星期六Five Classes of AntibodiesFive Classes o
8、f AntibodiesIgA 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.第9页，共21页，编辑于2022年，星期六Antibody ValidationAntibody ValidationStep 1:Show reactivity on a Western(denatured epitope).Step
9、 2:Show IP ability.Perform IP,run Western and re-probe with same Ab(IP-Western)Step 3:Rough quantification,true band should be 50%of combined signal from other bands(ENCODE guidelines)第10页，共21页，编辑于2022年，星期六Positive and Negative AbsPositive and Negative AbsPositivePositive 1 Ab=anti-wheat germ 1 Ab=a
10、nti-wheat germ RNA PolII;murine MAb RNA PolII;murine MAb(DAY 1)(DAY 1)2 Ab=Rabbit Anti-Mouse IgG2 Ab=Rabbit Anti-Mouse IgG (DAY 2)(DAY 2)NegativeNegative 1 Ab=IgG pool from 1 Ab=IgG pool from rabbit serum(nonspecific,rabbit serum(nonspecific,cheap)(DAY 1)cheap)(DAY 1)Addition of 2 Ab to match Additi
11、on of 2 Ab to match the samples(anti mouse is the samples(anti mouse is non-reactive to rabbit IgG),non-reactive to rabbit IgG),some protocols will omit some protocols will omit(DAY 2)(DAY 2)Staph A binds more efficiently to Rabbit IgG第11页，共21页，编辑于2022年，星期六Workshop Antibodies and DNA Workshop Antibo
12、dies and DNA Positive Control:mouse anti-PolII primary with rabbit anti-mouse IgG secondaryNegative Control:Nonspecific Rabbit IgG with rabbit anti-mouse IgG secondaryThe supernatant from the Negative Control will be used as“Input”later!1222第12页，共21页，编辑于2022年，星期六StaphylococcusStaphylococcus aureusau
13、reus binding binding (DAY 2)(DAY 2)WashesProtein A protects StaphA from host defenses第13页，共21页，编辑于2022年，星期六Elution and Crosslink Reversal Elution and Crosslink Reversal 1)Addition of NaHCO3 causes antibodies to release transcription factors(DAY 2)2)0.2 M NaCl at 67 for 3 hours or more reverses cross
14、links,DNA fragments now free in solution(DAY 2)3)RNase A is added digest RNA in sample(DAY 3)4)Qiaquick PCR Purification kit is used to purify DNA for PCR,comparing positive Ab,negative Ab,and Input(DAY 3)第14页，共21页，编辑于2022年，星期六PCR to Verify ChIP(DAY 3)PolII PrimersDHFR3 UTR PrimersVerify that:1)ChIP
15、 of interest(PolII for us)is greater for target promoter compared to Input vs.negative control compared to Input2)Positive Ab ChIP(polII)is much higher than negative Ab ChIP(IgG)for target promoter compared to negative controlRemember that if you are doing ChIP on Chip,you will be using the positive
16、 Ab ChIP samples and the Input as labeled targets,NOT the negative ChIP(IgG)Lane Sample1.MW2.1 ul polII ChIP3.1 ul IgG ChIP4.1 ul 0.004%Input5.1 ul 0.02%Input6.1 ul 0.2%Input7.1 ul polII ChIP8.1 ul IgG ChIP9.1 ul 0.004%Input10.1 ul 0.02%Input11.1 ul 0.2%Input12.12.MW第15页，共21页，编辑于2022年，星期六Sonicated C
17、hromatin100 ul(eg)100%InputNo Ab(or IgG supernatant)10 ul Input+90 ul Buffer=10%Input+AbIPed,washed,elutedReverse crosslinksQIAquick purifyElute in 50 ul EBSonicated Chromatin 100 ul(eg)100%Input10%Input/50 ul=0.2%Input/ulPositive Ab ChIP samplePCR(positive control)1 ul0.2%1 ul 1:100.02%1 ul 1:500.0
18、04%1 ul1 ul=0.02%50 ul=1%of InputAim for 0.2-2%Efficiency of ChIP第16页，共21页，编辑于2022年，星期六WGAChIPInputLabel w/Cy DyesApply to microarrayCompare Red/Green signal intensities to identify binding sitesPeak Finding a challenge,but commercial software and local tools becoming more availableChIP-chip第17页，共21
19、页，编辑于2022年，星期六ChIP-Seq第18页，共21页，编辑于2022年，星期六Whole Genome AmplificationPrinciple of procedurechIP samples start hereMethod ComparisonInput DNAGel analysis of products第19页，共21页，编辑于2022年，星期六AcknowledgementsAcknowledgements Farnham labFarnham labProtocol DevelopmentProtocol DevelopmentShally XuShally Xu
20、Sharon SquazzoSharon SquazzoCells&MaterialsCells&MaterialsAlina RabinovichAlina RabinovichShally XuShally XuWGA DataWGA DataHenny OGeenHenny OGeen第20页，共21页，编辑于2022年，星期六100 ul Sonicated Chromatin1,000,000 promotersNo Ab(or IgG supernatant)10 ul Input+90 ul Buffer=100,000 promoters+AbIPed,washed,elute
21、dReverse crosslinksQIAquick purifyElute in 50 ul EB100 ul Sonicated Chromatin1,000,000 promoters100,000 promoters/50 ul=2,000 promoters/ulPositive Ab ChIP sample?promoters/ulPCR(positive control)1 ul2,000 promoters1 ul 1:10200 promoters1 ul 1:5040 promoters1 ul1 ul=200 promoters50 ul=10,000 promotersAim for 2,000(0.2%)or betterEfficiency of ChIP第21页，共21页，编辑于2022年，星期六