啟動(dòng)子活性分析課件

1、啟動(dòng)子活性分析基因表達(dá)調(diào)控的研究策略和常用方法 2015-9-22 基因表達(dá)調(diào)控是多層次的復(fù)雜過(guò)程啟動(dòng)子（Promoters） 是位于結(jié)構(gòu)基因5端上游的DNA序列，其長(zhǎng)度從100 bp到200 bp不等，與轉(zhuǎn)錄起始時(shí)RNA聚合酶識(shí)別、結(jié)合和啟動(dòng)轉(zhuǎn)錄有關(guān)。啟動(dòng)子控制基因轉(zhuǎn)錄的起始時(shí)間和表達(dá)的程度。啟動(dòng)子的組成promoter普遍轉(zhuǎn)錄因子啟動(dòng)子活性分析 Reporter Assay EMSA ChIP（一）氯霉素乙酰轉(zhuǎn)移酶基因（CAT）可催化乙酰CoA的乙?；D(zhuǎn)移到氯霉素3羥基，而使氯霉素解毒。線性范圍較窄，靈敏性較低 （二）半乳糖苷酶 由大腸桿菌lacZ基因編碼，可催化半乳糖苷水解。 （三） 熒

2、光素酶（Luciferase） 是自然界中能夠產(chǎn)生生物熒光的酶的統(tǒng)稱。熒光的產(chǎn)生是來(lái)自于熒光素（底物）的氧化，哺乳細(xì)胞無(wú)內(nèi)源性熒光素酶。 最常用的熒光素酶有細(xì)菌熒光素酶、螢火蟲熒光素酶和Renilla熒光素酶。螢火蟲熒光素酶靈敏度高，檢測(cè)線性范圍寬，是最常用于哺乳細(xì)胞的報(bào)告基因。（四）熒光蛋白家族：GFP, BFP,RFP promoterluc缺失（deletion）突變（mutation）熒光素酶報(bào)告基因檢測(cè)Gel Shift AssayElectrophoretical Mobility Shift AssayEMSABand Shift Assay1. Introduction2. P

3、rinciple3. Application4. Materials5. Procedure6. Proof of SpecificityContentsDNA-protein complexes migrate slower than non-bound DNA in a native polyacrylamide or agarose gel, resulting in a “shift” in migration of the labeled DNA band.PrincipleR. Voll 09/01A double-stranded oligonucleotide containi

4、g a NF-B- binding site is labeled with a radioactive isotope and incubated with different nuclear extract.During gel-electrophoresis, NF-B bound to the oligonucleotide causes a shift compared to the free probe.NF-BFree ProbeRadioactively labeled oligonucleotidewith NF-B - binding site (probe) and bo

5、und NF-B complexesRadioactively labeled oligonucleotide with NF-B - binding site (probe)Nuclear extract of non-activated cellsNuclear extract ofactivated cellsApplicationDetection of DNA-binding factors/proteins1. Analysis of DNA sequences (e. g. promoter or enhancer regions) for their potential to

6、bind specifically to proteins/nuclear extracts 2. Analysis of (sub-)cellular extracts for the presence of certain DNA-binding proteins (e. g. a transcription factor with a known recognition sequence)Materials3 or 5 end-labeled DNA target.Protein extractunlabeled DNA targetPolyacrylamide gel in 0.5X

7、TBEElectrophoresis apparatusPositively charged nylon membraneElectroblotter transfer apparatusUV lampX-ray filmPrepare the ProbeR. Voll 09/01AP-1biotin-5-GCT TGA TGA CTC AGC CGG AA C-3 3-CGA ACT ACT GAG TCG GCC TT G-5-biotinNF-kBbiotin-5-AGT TGA GGG GAC TTT CCC AGG C-3 3-TCA ACT CCC CTC AAA GGG TCC

8、G-5-biotin Binding motif1. Synthesize complementary single stranded biotin-labeled and unlabeled oligonucleotides containing a transcription factor binding site.2. Annealing the Oligos：Heat up an equimolar mixture of the 2 oligos to 95C and let them slowly cool down by turning off the heat block.Pla

9、n Binding ReactionsR. Voll 09/01 Procedure1. Prepare nuclear protein extracts2. Prepare Biotin- labeled and unlabeled target DNA3. Plan Binding Reactions4. Prepare and Pre-Run Gel5. Prepare and Perform Binding Reactions6. Electrophorese Binding Reactions7. Electrophoretic Transfer of Binding Reactio

10、ns to Nylon Membrane8. Cross-link Transferred DNA to Membrane9. Detect Biotin-labeled DNA by ChemiluminescenceProof of SpecificityR. Voll 09/011. Supershift using antibodies against the DNA-binding protein ( protein Specificity )2. Competition for binding to the labeled probe using unlabeled wildtyp

11、e and mutated oligos ( DNA Specificity ) R. Voll 09/01Competition with Unlabeled OligosIncreasing amounts of unlabeled oligos containing the NF-kB binding siteor unlabeled oligos with a mutated binding site were added to the reaction mixprior to gel electrophoresis. Specific binding is extinguished only by the non-mutated oligo.Free probep50/p65Wild type oligo Mutated oligoGGG GAC TTT CCCGGA GAC TTT CCCChromatin immunoprecipitation Used to determine whether a given protein binds to a given DNA sequence in vivoLike all protein analysis involving anti