AKTA--實驗室層析柱裝柱技術(shù)介紹.ppt

實驗室層析柱裝柱技術(shù)介紹,Columnpacking,好的柱效是層析成功的關(guān)鍵不同種類的凝膠、不同的層析柱有不同的裝柱方法柱效測定的方法層析柱的維護,Columnpacking,RabiesvaccineSepharose4FastFlow5%columnvolsample,Columnpacking,每米塔板數(shù)：N=1600,每米塔板數(shù)：N=3500,Columnpacking,N/m=5.54(Ve/Vh)2100/L,As=b/a,Columnpacking,檢測裝柱效果一般用0.5%(30um介質(zhì))或1%(90um介質(zhì))柱體積的1%丙酮測柱效和峰形Thelinearflowrateshouldbe30cm/hfor34mand50mmediaand20cm/hfor90mmedia,2019/11/23,線性流速對體積不同的柱子來說，體積流速不能直接用作比較，一般都換算成線性流速，再作比較：體積流速(ml/min)x60線性流速(cm/hr)=揪揪揪揪揪揪柱子橫切面積(cm2),Columnpacking,Columnpacking,Itisimportantthatthecolumnisproperlyequilibratedbeforethepackingisevaluated(2columnvolumes).Itispreferabletorunthreetestrunstoseewhetherthetestvaluesarestable.Ifapoorresultimprovesduringthistest,thereasoncanbethatthecolumnwasnotproperlyequilibrated.Tocheckthatthebedisstable,runthecolumnat70%packingpressurefor20hoursandtestitagain(preferablythreetestruns).Notethatpressurespikesmaycausepoorpacking(cracking).Ifthishappens,anairtrapandapressurereliefvalveareneededbetweenthepumpandthecolumn.Thepressurereliefvalveshouldbeplacedbetweentheairtrapandcolumn.,Columnpacking,裝柱注意點,選擇一根設(shè)計良好的柱子仔細混合均勻凝膠漿在生產(chǎn)時使用柱子的溫度下裝柱用平緩的動作將凝膠漿一次性加入柱子中用恒壓或恒流裝柱穩(wěn)定和平衡凝膠柱床檢查柱效，必要時重裝,Columnpacking,2019/11/23,用空柱子須留意.,使用調(diào)節(jié)器(flowadaptor)-保護柱床的同時更可提高分辨率，節(jié)省凝膠柱子物料須生物兼容，并有優(yōu)良的化學抗性加上裝柱器(packingreservoir)，可一次把凝膠裝滿柱子，大大提高柱效，尤其是凝膠過濾柱。柱子的過濾網(wǎng)孔徑應該是凝膠顆粒的1/3,一般10mm孔徑就夠用，如用SOURCE15介質(zhì)裝C柱和XK柱，須換上5mm的網(wǎng),Columnpacking,SepharoseFFbasedgel:SepharoseQ,DEAE,SP,Phenyl1.the75%slurryjustbeforepacking.2.Ideally,FastFlowmediaarepackedataconstantpressurenotexceeding3bar(0.3MPa)forXKcolumns.3.Ifthepackingequipmentdoesnotincludeapressuregauge,useapackingflowrateofatleast400cm/h(15cmbedheight,25).Ifrecommendedpressureorflowratecannotbeobrained,usethemaximumflowthepumpcandeliver,thisshouldalsogiveaeasonablywell-packedgel.Donotexceed75%ofthepackingflowrateinsubsequentchromatographicprocedures.,Columnpacking,Sepharose4FastFlow50-75%slurryjustbeforepacking.110cm/hflowrateandthepressureshouldnotbeincreasedbeyond1.0bar3-5mmblowthesettledbedwasmade,Columnpacking,SepharcylSHRFlowratesgivenbelow.PackthegelinSTEP1for2hoursoruntilthegelhasreachedaconstantheight,thenincreasetheflowratetothevaluelistedforSTEP2andpackfor60minutes.,Packingflowrates(ml/h):ColumnSTEP1STEP2XK16/7060110XK16/10060100XK26/70150300XK26/100150270XK50/100500800,Columnpacking,Superdex75,200pg50%slurry1step:20cm/hflowrate,2hour2step:stepthepressureisheldat3bar,1hour,Columnpacking,SephadexG-2550%slurry400cm/hflowrate3mmblowthesettledbedwasmade,層析柱的使用及維護,柱的平衡：在裝柱及做完一個層析后，用至少5倍柱體積的緩沖液平衡或柱的流出液的電導和pH穩(wěn)定。柱的再生：用高離子強度的緩沖液（1MNaCl)洗脫或改變pH。,在位清洗（CIP）,離子性吸附的蛋白:Washwith0.5CV2MNaCl.Contacttime15min.沉淀的、疏水性結(jié)合的蛋白及脂蛋白：1MNaClat40cm/h,1-2hours.脂類及非常強結(jié)合的疏水性蛋白：2-4CV0.5%非離子去垢劑（或1%醋酸）1-2小時。使用梯度方式70%的乙醇或30%的異丙醇，2-4CV1-2小時。,柱子的保存,0.15mol/LNaAcpH4.020%ethanol0.01mol/LNaOH0.05%sodiumazide,