细胞与分子生物学

2006.1PCRELISA2008.6834544DNS-44RNA55PCRPCR5PCR4PCR43ELISAWesternblot63638/3349ß-actin201DNA2PCRß-actin3DNA4southernDNAAKPKm201AKP2AKP3DEAEDE-52AKP4AKPKm912132DNS-RNAPCRPCRPCRPCRELISA/ß-actinDNAPCRß-actinDNAsouthernDNAAKPKmAKPAKPDEAEDE-52AKPAKPKm1.2.3.1.2.3.4.5.6.1.2.pH3.4.5.1.2.3.1.2.(Electrophoresis)50(Zeta)FQEF=QEFStokerF=6rvFFQE=6rv(mobility)=v/E=Q/6r=v/E=(d/t)/(v/l)=dl/vtv()E()d()l()u()1()()2-1-112dl=lVt/ld2=2Vt/l(V)t()d=d1d2=(12)Vt/l124()pHpHpHpH20pH55pH(Q)pHpHpH86(Tris)()pH(Zeta)002-02I=CiZi2/2Ci=Zi=1(NaCl)0154molLNaCII=(0.154I20.154I2)/2=0.1542(ZnSO4)401molLZnSO4I=(0.1220.122)/2=0.4()1515010(50)()()()(agaropetin)pH()1()234()1234(discelectrophoresis)2550um50100cm()2-3()pH1pHpH2.pHpHpHpH(Electrofocusing)pH(Ampholin)pHIsotachophoresis)pH()()()()Kohn(1975)()081pH6-9002-0056512DNADNADNA%Agarose0.30.60.70.91.21.52.05-60kb1-20kb0.8-10kb0.5-7kb0.4-6kb0.2-4kb0.1-3kb()(polyacrylamidegeldectrophoresisPAGE)(1100)(SDS)1(Act)(Bis)27550A3020A%1000010000-4000040000-100000100000-50000050000020-3015-2010-155-102-51000010000-100000100000-200000015-205-102-2.63pH(pH9)pH(pH4)pHpHpHpH94)00101molL4()(1)TrisHClpH67TrisHClpH89TrispH83pHHClC1-NH2CH2COO-(Cl-)(NH2CH2COO-)(2)(3)pHpH89(95)pKa(9798)pH5SDS(SDS)8X1041'1'10VL114SDSSDSSDS(2)09SDS14SDS6pHpH001pHpHpH(1)pH(2)pH(3)(4)(5)60SvessonVesterbergpHAmpholine()RlR2pH3102080()1975OFarrellSDS1,10003125(35S)x1.2.3.4.5.1.2.3.4.1.2.pI75pH86A12()12pH860061276g168g1000ml310B10B05g50ml10ml40ml10B(aminoblackl0B)C22H13O12N6S3Na3MW=715,max=620-630nmSDS()-4.95%45ml5ml50ml5S()631(1)2x8cm20cra(2)()(3)()(4)35ul25452()510Vcm()0406mAcm310B(S)54604mol/L4ml650nm1,2,,5T=A+1+2++%=A/T100%1%=1/T100%2%=2/T100%=/T100%=/T100%95(28)2015772125249651212202232157?2?3?1.C2.Ampholine3.pHpI4.1.2.3.4.1.2.pIpHpHpHpHpHpHpH3001000Ampholine(LKB)Pharmalyte(Pharmacia)pHpHpHpHpHpHpHpHAmpholinepHpIpHAmpholinepIpH001pI6.9CpI05pI1()002molLNaOH()001molLH3PO4220(Ampholine)3C430300NN08100ml451061TEMED71(1)1005cm()7cm(2)mlAcrBis40%Ampholinemg/ml10%2.00.24(HbCytC)7.00.159.35ml(3)7ml(4)5ml6.5510cm05cm202H2SO45H3PO4042NaOH50V30200V300V123(10cm)4pI(1)5ml4pHpI(2)pH1?2?3?1.d-3.6--l-2.3.4.1.2.3.4.5.1.2.B6-12345BB0.1g10ml37615.4g2.76gEDTA0.29g1000mlpH8.60.07570.5%0.5g100ml10.2ml0.02ml37302000/520.5%50603ml1.5330L4.120V34mA2/35.-+-++-+---1.2.373.4.5.55%15%30%CM1.1.M.Tswett2.3.1234.1.2.3.1.2.RfRf=/(100)VAVGMVi()VO()(Sephadex)(BioGel)(Sepharose)(Dextran)A(A)VOCVOViBVeVe=VO+KdViKdKd=(Ve-VO)ViKd=1Ve=VO+ViKd=0Ve=VO0Kd1Ve=VO+KdViKdtABCViVi=aWra=Wi=a=Vt(ml)/ml/gVt=Vo+Vi+VgVg=Vt=Vt=14D2hh//20-2590-100G-10-700-7001.0231G-15-1500-15001.5331G-25100-50001000-50002.5531G-50500-100001500-300005.01031G-751000-5003000-8009.512-152430000G-1001000-1000004000-15000010.015-20725G-1501000-1500005000-40000015.020-30725G-2001000-2000005000-80000020.030-40725()12(1)234a)X+.YH+Z+2YH+Z+X+3YH+4X+Z+R-X++Y+R-Y++X+R-X+Y+1pHpH2()(RSO3H)RS03H+Na+RSO3HNa+H+(PO3H2)(PO2H2)(COOH)(OH)RCOOH+Na+RCOONa+H+()N+(CH3)3RN(CH3)3OH+Cl-RN(CH3)3Cl+OH-N(CH3)2NHCH3NH2()RNADNA[OligedTPoly(A+)RNA()()Sephrose2B4B6B(BrCN)()RfRf=/(pH)RfRf()90b)c)d)10-100e)-GG(HPLC)34X107530HorvathKirklandHPLC(5um)HPLC1.2.3.Kd4.5.Folim-denis1.2.3.Kd4.1.2.(64500)(DNP200012000)G-50(Sephadex)Kd1.G-502.3.24.25.16.17.18.19.10.11.09NaCl12.13.10NaHC0314.15.95(1)(Hb)2ml509NaCl5Hb(2)DNP--1510NaHC0315rnl(pH85-90)159530ml595DNP59510mlDNP(3)Hb3miDNP5mi108-1.2cm25cm2-3mlG-5023em18cm()512152.()4123()VeVo()Ve4(Vt)(Vo)(Vi)(Ve)Hb(Kd)5101231?2?3.kd=0kd=075(1)?(2)?DNS-1.DNS-2.6-103.1.2.3.DNS-1.DNS-2.RfRf=/(1Dimethylaminonaphthalene5SulfonylchlorideDNSCl)DNSDNSDNS10-9~10-101066DNS-1.2.3.4.5.6.7(4x4cm)89.pH(pHl-14)10.5mg20mg40mg50mg10ml02mollNaHCO21102molLNaHCO3NaOHpH9812DNS-(AR)DNSCl25mgml13(1)91(V/V)(2)15100(V/V)141molLHCl1molLNaOH1DNS5DNSCl44030lmolLHCIpH2-3(pH)pH23lmolLNaOH67DNS-2DNS-(1)44cmlcmNDS23mm(2)2mm(3)(4)90(5)DNSRfI-II-DNS12341??2?3DNSRf?4?1.2.pH3.1.2.3.4.1.2.pHpIpHpIpIpHDowex50AspGlyLysp12775959.74pH42Dowex501(0812cm)X25cm234567217pH()801molLNaOH15NaOH96molLHCl10006molLpH422H2O882g42ml12molLHCl6ml80()5000mlpH4211GlyAspLysl0mg30ml006molLpH421290ml180ml3306ml26161224H2O0369ml90ml13Dowex50Dowex506molHCI1HCIpH15NaOH1NaOHpHpH42123ml2Dowex50Dowex50Dowex501820cm301molLNaOH6pH4.210pHpH42152045()pH42pH42512102ml(40)15570mm01molLNaOH601molLNaOH6701mol/LNaOH8(-+++…)12AspGlyLys?1.2.3.r/mingxg4.5.1.2.1.2.3.r(g)1500021Centrifugalforce(F)F=m2r()r:(cm)m2Relativecentrifugalforce(RCF)RCFRCF=FF=m2r/mg=2r/gg(98070g/sec2)2(nr/min)=2xn/60(rad/sec)RCF=(2xn/60)2/98070r=1.11810-5n2rrming(Xg)XgDole&Cotzias3Sedimentationcoefficient(S)SS=/=(dr/dt)/r2SS10-1310-13SvedbergS1S110-13S()1.6000r/min225000rmin385000-min(600000g)DNARNA()(fixedanglerotor)(swingoutrotor)(verticalrotor)(zonalrotor)(continuousrotor)11440202834402903901.(differentialvelocitycentrifugation)(ratezonalcentrifugation)2(isopyniccentrifugation)3()2()1(KBrCsCl)2123(4)(5)()1drdt2(1)(2)345()12pH3456()1133g/mlRNA2FicollFicoll400400000Ficoll3191gndDNA4KBrNaCIKINaIRNANaClNaIDNA5Percoll:SiO2(PVP)pH(DNA)RNA1.2.3.RNA1.2.RNA3.1.2.1500g10min,10000g10min,100000g3h,2RNAm-RNA,TrizolRNA