L.HEREDITAS(Beijing)28(6):721 725,2006/ZEl:2005 06 18;:2006 02 13:SE1S(I|:39870078, 30370128, 30570146) Supported by the National Natural Science Foundationof China(No.39870078, 30370128, 30570146)Te:lS(1970),3,pV3,=,Z_:y。E-mail:qixiaotingmail.cnu.edu.cnYT:v2(1960),3,q,pV3=,Z_:y。E-mail:tychaigucas.ac.cn;Tel:010-88256343/S:DNA_k4ELZE祁小廷1,2 , 柴小清2 , 刘靖2 ,柴团耀1(1.SS33,100049;2.n=Sv3S,100037)K1:EL(gel retardation),%MqML(Electrophoreticmobilityshift assay,EMSA),DNAMTB/。.d32PS:EL,2,79(BbOs。KdbS:EL,X,ZEy,2,dbS:Ek4n。,4B/S:DNA_k4ELZE。n5|DNAEcoRTL3S:,N1LRS:DNA_k4(DIGHighPrimeDNA Labeling andDetectionStarter Kit , Rohe)S:E|_。VQL,KT,S:DNA_k4EL40ZE。1oM:EL;ms|:Q75DSM:AcI|:0253-9772(2006)06-0721-05A New Method for EMSA by ModifyingDIGHighPrime DNA Labelingand DetectionStarter Kit QI Xiao-Ting1,2 , CHAI Xiao-Qing2 , LIUJing2 , CHAITuan-Yao1(1.Department of Biology, GraduateUniversityof ChineseAcademy ofSciences, Beijing 100049, China;2.CollegeofLifeScience , CapitalNormal University, Beijing 100037, China)Abstract:Gel retardation, also named electrophoretic mobility shift assay(EMSA) , isa useful tool for identifyingprotein-DNA interactions. Typically, 32P-labeled DNA probes used in EMSA are sensitive. However, it reliesonthehandlingofhazardousradioisotopes, and isnoteasilyquantified. Recently, somesuccessful caseshavebeenreportedusingnon-radio labelled probesinsteadofradiolabelled probesinEMSA. The method israpid, convenient, and safe,but itdependsona veryexpensive kit. In thisstudy, we offered a new method performing EMSA by modifying DIGHighPrime DNALabelingandDetectionStarter Kit(Rohe). Firstly, the preparedlabeledprobe wasintroducedtheEcoR stick intheendofprobefor3-endlabeling, andthenwasperformed the probe labeling anddetecting thesig-nalsof EMSAwiththerelativelycheapDIGHighPrimeDNALabelingandDetectionStarter Kit(Rohe). Byadjustingthe experiment parameters, thesuccessful resultwasobtained. Thepresent study provides asuccessful example andmethodfor modifying DIGHighPrimeDNA Labeling and DetectionStarter Kit.Keywords:electrophoreticmobility shift assay(EMSA);DIG;probesEL(gel retardation),%MqML(electrophoretic mobility shift as-say),Be、y2_Y4|+sDNA/ 1 。y%4|+DNAs01l,1|kDNAs0S:,S:(digoxigenin,DIG)S:。.dELS:32Pb,y27$,+s%i134 bpDNA+sy0。m2V1ELTFig.2Electrophoretic mobility shift assay accordingto theparameters described in Table 1V2Q8(ldFDNA/=3/6.52/7.5)Table 2Binding reaction system with theratio(3/6.5or2/7.5)ofcalf thymus DNA to nuclear protein preparation3Parameter38sComponentsof binding reactionsystemvCKLFTestFCompetitor4 Parameter4vCKLFTestFCompetitor10A10buffer 1.5 1.5 1.5 1.5 1.5 1.5ldFDNA(1g/L)CalfthymusDNA 3.0 3.0 3.0 2.0 2.0 2.0S:(2ng/L)Labelled probes 1.0 1.0 1.0 1.0 1.0 1.0(1g/L) Nuclearprotein - 6.5 6.5 - 7.5 7.5DNA(80 ng/L)CompetitionDNA - - 3.0 - - 3.0)Sterile distilled water 9.5 3.0 - 10.5 3.0 -:L。Unit:L.m3V2ELTFig.3Electrophoretic mobility shift assay accordingto the parametersdescribed in Table23)S:DNA_k4SEL,Kv5DNAS:。DIGS:Ek4(DIGGel Shift Kit),S:1ZEM3FBDIG-11-ddUTP,V7P,rS:9 。k4S:DNAk4,YV/rS:134bpDNA(m4):1)SDNAVEcoRMl,BEcoRT;2)k4S:k4cKlenow724L.HEREDITAS (Beijing)2006285l,sDIG-dUTP,KXfs0S4DIGs0;3)S:zVPCRylk4V,rB。ZE,S:DNAs0,_(m1),ELT9VS:ZEV(m2 ,m3)m4DIGS:U