生理所黄阿敏课件

Southern,Northern and Western blotting生理所黃阿敏Comparison of Southern,Northern,and Western analyses of Gene XSouthern hybridizationnFirst described by E.M.Southern in 1975.nApplications of Southern hybridizationnRFLPs,VNTRs and DNA fingerprintingnChecking of the gene knockout micenThe flow chart of Southern hybridizationSouthern hybridizationTransfer bufferDetection of an RFLP by Southern blottingDetection of the sickle-cell globin gene by Southern blottingChecking of the gene knockout miceFlow chart of Southern hybridizationPreparing the samples and running the gelSouthern transferProbe preparationPrehybridizationHybridizationPost-hybridization washingSignal detectionIsotopeNon-isotopePreparing the samples and running the gelnDigest 10 pg to 10 g of desired DNA samples to completion.nPrepare an agarose gel,load samples(remember marker),and electrophorese.nStain gel ethidium bromide solution(0.5 g/ml).nPhotograph gel(with ruler).Critical parameters(I)nNote the complexity of DNAnGenomic DNAnA single-copy of mammalian gene,3 Kb average in length 10 g x 3 Kb/3 x 106 Kb=10 g x 1/106=10 pgnPlasmid DNA or PCR products 0.1 g of a 3 Kb plasmid DNA 100 ng Gel treatmentnAcid treatment n0.2 N HCl solution nDenaturationnNaOH solutionnNeutralizationnTris-Cl buffer(pH8.0)Southern transfernMeasure gel and set up transfer assembly:nWick in tray with 20 x SSCnGelnNitrocellulose or Nylon filters(soaked in H2O and 20 x SSC)n3MM Whatman filter papernPaper towelsnWeightAfter Southern transfernDissemble transfer pyramid and rinse nitrocellulose in 2x SSCnBake nitrocellulose at 80C for 2 hr or UV-crosslink Nylon membrane for seconds Preparation of probesnSynthesis of uniformly labeled double-stranded DNA probesnPreparation of single-stranded probesnLabeling the 5 and 3 termini of DNASynthesis of double-stranded DNA probes-Nick translation of DNA-Labeled DNA probes using random oligonucleotide primersNick translationPreparation of single-stranded probesnSynthesis of single-stranded DNA probes using bacteriophage M13 vectors.nSynthesis of RNA probes by in vitro transcription by bacteriophage DNA-dependent RNA polymerase.In vitro transcriptionnLabeling the 3 termini of double-stranded DNA using the Klenow fragment of E.coli DNA polymerase I.(lack of 5 3 exonuclease activity)nLabeling the 3 termini of double-stranded DNA using bacteriophage T4 DNA polymerase.nLabeling the 5 termini of DNA with bacteriophage T4 polynucleotide kinase.Labeling the 5 and 3 termini of DNAT4 polynucleotide kinase activityNon-isotope labelingnDigoxigenin-11-dUTP(DIG-dUTP)labeling-DNA labeling-Oligonucleotide labeling-RNA labelingPCR Labeling,Random Primed Labeling,and RNA LabelingPrehybridization nAdd prehybridization solution and prehybridize at hybridization temperature for 2-4 hr Hybridization nRemove prehybridization solution and add hybridization solutionnAdd 500,000 cpm of the probe/ml hybridization solution.nHybridize overnight at appropriate temperature.Post-hybridization washing n Wash twice,15 min each,in 1x SSC,0.1%SDS at room temperature.nWash twice,15 min each,in 0.25x SSC,0.1%SDS at hybridization tempCritical parameters(II)nHomology between the probe and the sequences being detectednTm=81+16.6(log Ci)+0.4%(G+C)-0.6(%formamide)-600/n-1.5(%mismatch)nFactors can be changed:nHybridization temp.nWashing temp.nSalt concentration during washingHigh temp.,low salt:high stringencyLow temp.,high salt:low stringencynIf 50%formamide is usedn42 oC for 95 100%homologyn37 oC for 90 95%homologyn32 oC for 85 90%homologyComparison of nitrocellulose and nylon membranesNC Nylon Hydrophobic binding Covalent binding Fragile Durable Probe length 200 300 bp 0.05%of the message),total cellular RNA can be used.If the mRNA species of interest is relatively rare,however,it is advisable to use poly(A)+RNA.nIncubate 15 min at 55CRunning the RNA gelnAdd 10 l formaldehyde loading buffer to each sample and load gel.Run gel at 100 to 120 V for 3hr.nRemove gel from the running tank and rinse several times in water.Place gel in 10 x SSC for 45 min.nDo not need post-transferring gel treatmentAn example of Northern blottingNorthern blotRNA gel 28 S 18 SWestern blotting,or immunoblottingTechnique for detecting specific proteins separated by electrophoresis by use of labeled antibodies.Flow chart of Western blottingElectrophoresing the protein sampleAssembling the Western blot sandwichTransferring proteins from gel to nitrocellulose paperStaining of transferred proteinsBlocking nonspecific antibody sites on the nitrocellulose paperProbing electroblotted proteins with primary antibodyWashing away nonspecifically bound primary antibodyDetecting bound antibody by horseradish peroxidase-anti-Ig conjugate and formation of a diaminobenzidine(DAB)precipitatePhotographing the immunoblotSDS polyacrylamide-gel electrophoresis(SDS-PAGE)Analysis of protein samples by SDS polyacrylamide-gel electrophoresis and Western blottingProtein bands detected by specific antibodySDS-PAGEWestern blotComparison of Southern,Northern,and Western blotting techniques