AKTA--实验室层析柱装柱技术介绍.ppt

实验室层析柱装柱技术介绍,Columnpacking,好的柱效是层析成功的关键不同种类的凝胶、不同的层析柱有不同的装柱方法柱效测定的方法层析柱的维护,Columnpacking,RabiesvaccineSepharose4FastFlow5%columnvolsample,Columnpacking,每米塔板数：N=1600,每米塔板数：N=3500,Columnpacking,N/m=5.54(Ve/Vh)2100/L,As=b/a,Columnpacking,检测装柱效果一般用0.5%(30um介质)或1%(90um介质)柱体积的1%丙酮测柱效和峰形Thelinearflowrateshouldbe30cm/hfor34mand50mmediaand20cm/hfor90mmedia,2019/11/23,线性流速对体积不同的柱子来说，体积流速不能直接用作比较，一般都换算成线性流速，再作比较：体积流速(ml/min)x60线性流速(cm/hr)=揪揪揪揪揪揪柱子横切面积(cm2),Columnpacking,Columnpacking,Itisimportantthatthecolumnisproperlyequilibratedbeforethepackingisevaluated(2columnvolumes).Itispreferabletorunthreetestrunstoseewhetherthetestvaluesarestable.Ifapoorresultimprovesduringthistest,thereasoncanbethatthecolumnwasnotproperlyequilibrated.Tocheckthatthebedisstable,runthecolumnat70%packingpressurefor20hoursandtestitagain(preferablythreetestruns).Notethatpressurespikesmaycausepoorpacking(cracking).Ifthishappens,anairtrapandapressurereliefvalveareneededbetweenthepumpandthecolumn.Thepressurereliefvalveshouldbeplacedbetweentheairtrapandcolumn.,Columnpacking,装柱注意点,选择一根设计良好的柱子仔细混合均匀凝胶浆在生产时使用柱子的温度下装柱用平缓的动作将凝胶浆一次性加入柱子中用恒压或恒流装柱稳定和平衡凝胶柱床检查柱效，必要时重装,Columnpacking,2019/11/23,用空柱子须留意.,使用调节器(flowadaptor)-保护柱床的同时更可提高分辨率，节省凝胶柱子物料须生物兼容，并有优良的化学抗性加上装柱器(packingreservoir)，可一次把凝胶装满柱子，大大提高柱效，尤其是凝胶过滤柱。柱子的过滤网孔径应该是凝胶颗粒的1/3,一般10mm孔径就够用，如用SOURCE15介质装C柱和XK柱，须换上5mm的网,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,层析柱的使用及维护,柱的平衡：在装柱及做完一个层析后，用至少5倍柱体积的缓冲液平衡或柱的流出液的电导和pH稳定。柱的再生：用高离子强度的缓冲液（1MNaCl)洗脱或改变pH。,在位清洗（CIP）,离子性吸附的蛋白:Washwith0.5CV2MNaCl.Contacttime15min.沉淀的、疏水性结合的蛋白及脂蛋白：1MNaClat40cm/h,1-2hours.脂类及非常强结合的疏水性蛋白：2-4CV0.5%非离子去垢剂（或1%醋酸）1-2小时。使用梯度方式70%的乙醇或30%的异丙醇，2-4CV1-2小时。,柱子的保存,0.15mol/LNaAcpH4.020%ethanol0.01mol/LNaOH0.05%sodiumazide,