染色质免疫沉淀精.ppt

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1、染色染色质质免疫沉淀免疫沉淀第1页，本讲稿共21页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，本讲稿共21页Starting ChIPStarting ChIP Treat cells with form

2、aldehyde: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 CellsTissuesAnatomi

3、cal structuresSee protocol and web page for additional information on cross-linking and example protocols第3页，本讲稿共21页Formaldehyde CrosslinkingProtein-ProteinDNA-DNAOther Options第4页，本讲稿共21页 DouncingvDounce contains two sizes A is large clearance B is small clearancevForce of moving rod B in dounce res

4、ults: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，本讲稿共21页Sonication using the BioRuptor!Sonication using the BioRuptor!Increases reproducibility betwe

5、en samples Decreases contamination Easy第6页，本讲稿共21页Sonication CheckSonication CheckSonication results in many different sized fragments and should be verified for your experiment 6 pulses11 pulses21 pulses31 pulses500 bp10 kbp3 kbpFragment size limits for Qiaquick PCR Purification Column is 100 bp-10

6、 kb.100 bp第7页，本讲稿共21页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 heavy chain can The heavy chain can come in several different come in

7、 several different classes.classes.Hundreds of Abs to Hundreds of Abs to transcription factors are transcription factors are commercially availablecommercially available第8页，本讲稿共21页Five Classes of AntibodiesFive Classes of AntibodiesIgA about 15%of total antibody count.IgM makes up 10%of our total an

8、tibodies.IgD less than 1%IgE less than 1%IgG approx.75%of our serum Ig pool,most polyclonals and mAbs.第9页，本讲稿共21页Antibody ValidationAntibody ValidationStep 1:Show reactivity on a Western(denatured epitope).Step 2:Show IP ability.Perform IP,run Western and re-probe with same Ab(IP-Western)Step 3:Roug

9、h quantification,true band should be 50%of combined signal from other bands(ENCODE guidelines)第10页，本讲稿共21页Positive and Negative AbsPositive and Negative AbsPositivePositive 1 Ab=anti-wheat germ 1 Ab=anti-wheat germ RNA PolII;murine MAb RNA PolII;murine MAb(DAY 1)(DAY 1)2 Ab=Rabbit Anti-Mouse 2 Ab=Ra

10、bbit Anti-Mouse IgGIgG(DAY 2)(DAY 2)NegativeNegative 1 Ab=IgG pool from rabbit 1 Ab=IgG pool from rabbit serum(nonspecific,cheap)serum(nonspecific,cheap)(DAY 1)(DAY 1)Addition of 2 Ab to match the Addition of 2 Ab to match the samples(anti mouse is non-samples(anti mouse is non-reactive to rabbit Ig

11、G),some reactive to rabbit IgG),some protocols will omit(DAY 2)protocols will omit(DAY 2)Staph A binds more efficiently to Rabbit IgG第11页，本讲稿共21页Workshop Antibodies and DNA Workshop Antibodies and DNA Positive Control:mouse anti-PolII primary with rabbit anti-mouse IgG secondaryNegative Control:Nons

12、pecific Rabbit IgG with rabbit anti-mouse IgG secondaryThe supernatant from the Negative Control will be used as“Input”later!1222第12页，本讲稿共21页StaphylococcusStaphylococcus aureusaureus binding binding (DAY 2)(DAY 2)WashesProtein A protects StaphA from host defenses第13页，本讲稿共21页Elution and Crosslink Rev

13、ersal 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 crosslinks,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

14、 to purify DNA for PCR,comparing positive Ab,negative Ab,and Input(DAY 3)第14页，本讲稿共21页PCR to Verify ChIP(DAY 3)PolII PrimersDHFR3 UTR PrimersVerify that:1)ChIP of interest(PolII for us)is greater for target promoter compared to Input vs.negative control compared to Input2)Positive Ab ChIP(polII)is mu

15、ch 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 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

16、.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，本讲稿共21页Sonicated Chromatin100 ul(eg)100%InputNo Ab(or IgG supernatant)10 ul Input+90 ul Buffer=10%Input+AbIPed,washed,elutedReverse crosslinksQIAquick purifyElute in 50 u

17、l 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 1:500.004%1 ul1 ul=0.02%50 ul=1%of InputAim for 0.2-2%Efficiency of ChIP第16页，本讲稿共21页WGAChIPInputLabel w/Cy DyesApply to microarrayCompare Red/Green signal inte

18、nsities to identify binding sitesPeak Finding a challenge,but commercial software and local tools becoming more availableChIP-chip第17页，本讲稿共21页ChIP-Seq第18页，本讲稿共21页Whole Genome AmplificationPrinciple of procedurechIP samples start hereMethod ComparisonInput DNAGel analysis of products第19页，本讲稿共21页Ackno

19、wledgementsAcknowledgements Farnham labFarnham labProtocol DevelopmentProtocol DevelopmentShally XuShally XuSharon SquazzoSharon SquazzoCells&MaterialsCells&MaterialsAlina RabinovichAlina RabinovichShally XuShally XuWGA DataWGA DataHenny OGeenHenny OGeen第20页，本讲稿共21页100 ul Sonicated Chromatin1,000,00

20、0 promotersNo Ab(or IgG supernatant)10 ul Input+90 ul Buffer=100,000 promoters+AbIPed,washed,elutedReverse 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，本讲稿共21页