APT第二步实验阳离子纯化0125.docx

1、APT第二步实验阳离子纯化0125APT-第二步实验-阳离子纯化1、MMC时间：20150114层析柱规格16*20介质名称MMC Diamond层析介质量30.8ml样品名称DEAE (6HP)20150113 5峰上样量约32mg样品处理用Hcl调PH至6.0上样体积45ml程序如下：Time(min)BFlow(ml/min)Fracton(min/管）002245022465221708022BufferA：0.02mol/L PH 6.0 Tris-Hcl+0.1mol/L NaclBufferB：0.02mol/L PH 6.0 Tris-Hcl+1mol/L Nacl图谱如下：效

2、价检测结果如下：样品名称10ug聚集率%2ug聚集率%1ug聚集率%粗毒30ug/18.53100/空白84.56/DEAE20150113上样液，0.506mg/ml33.03/73.77/DEAE/MMC20150114流穿，0.43mg/ml70.09/DEAE/MMC2015011438-50t，0.063mg/ml76.07/DEAE/MMC2015011458-64t，0.081mg/ml73.46/DEAE/MMC2015011465-71t，0.080mg/ml77.4/结果显示：收集峰与流穿峰均无效价。时间：20150115层析柱规格16*20介质名称MMC Diamond层

3、析介质量28.6ml样品名称DEAE (6HP)20150113 6峰上样量约21mg样品处理用HAC调PH至4.5上样体积40ml程序如下：Time(min)BFlow(ml/min)Fracton(min/管）001.536001.536151.53190801.53BufferA：0.02mol/L PH 4.5 NaAC-HAC+0.1mol/L NaclBufferB：0.02mol/L PH 4.5 NaAC-HAC+1mol/L NaclMMC层析20150115，MMC样品0.483mg/ml-4.94絮团状物出现20150115，MMC样品10-32t，0.165mg/ml8

4、4.1920150115，MMC样品38-53t，0.131mg/ml86.6220150115，MMC样品55-70t，0.138mg/ml时间：20150116样品名称DEAE (6HP)20150113 6峰图谱如下：MMC层析20150116，MMC上样液0.626mg/ml1.63絮团状物出现20150116，MMC原样0.616mg/ml-2.9120150116，MMC-8-14t0.186mg/ml97.3820150116，MMC-15-19t0.161mg/ml77.5720150116，MMC-20-25t0.164mg/ml87.520150116，MMC-26-32t

5、0.140mg/ml10020150116，MMC-35-41t0.146mg/ml92.562、CM-FF时间：20150115层析柱规格16*20介质名称CM Bestarose FF层析介质量24ml样品名称DEAE (6HP)20150113 4-5峰上样量31.8mg样品处理用G-25柱交换HAC使 PH至4.5上样体积55ml程序如下：Time(min)BFlow(ml/min)Fracton(min/管）001.536001.536151.53190801.53BufferA：0.02mol/L PH 4.5 NaAC-HACBufferB：0.02mol/L PH 4.5 Na

6、AC-HAC+1mol/L Nacl图谱如下：CM层析（DEAE-4,5峰上样）20150115，CM上样液D-G25,0.356mg/ml2.33；4.01絮团状物出现20150115，CM流穿8-26t,0.129mg/ml-26.9720150115，CM31-33t,0.104mg/ml78.820150115，CM34-36t,0.230mg/ml48.6220150115，CM37-42t,0.145mg/ml-7.28絮团状物出现20150115，CM43-53t,0.252mg/ml0.6220150115，CM54-58t,0.225mg/ml92.16空白84.42对照，2

7、0120409,0.102mg/ml5.50%/2ug时间：20150117样品名称DEAE (6HP)20150113 5峰上样量37.8mg样品处理用HAC调PH至4.520150117，CM层析20150117，CM原液，峰50.,639mg/ml71.75过G-25后上样液0.417mg/ml80.18FT0.126mg/ml95.2720150117，CM-31-36t0.228mg/ml-15.92絮团状物出现20150117，CM-37-40t0.129mg/ml3.9720150117，CM-41-42t0.133mg/ml020150117，CM-43-46t0.296mg/

8、ml10.3857.53%/2ug20150117，CM-47-52t0.189mg/ml43.0620150117，CM-53-59t0.648mg/ml62.6120150117，CM-64-67t0.109mg/ml5.2690.20/2ug20150117，CM-47-52t，复测0.084mg/ml1002.0/2ug20150117，CM-43-46t，复测0.054mg/ml11.05/大团4.9%/2ug，大团状物出现3、SP-FF时间：20150116层析柱规格16*20介质名称SP Bestarose FF层析介质量26.6ml样品名称DEAE (6HP)20150113

9、6峰上样量28.7mg样品处理用G-25柱交换HAC使 PH至4.5（实际3.7）上样体积40ml程序如下：Time(min)BFlow(ml/min)Fracton(min/管）001.536001.536151.53190801.53BufferA：0.02mol/L PH 4.5 NaAC-HACBufferB：0.02mol/L PH 4.5 NaAC-HAC+1mol/L Nacl图谱如下：样品名称10ug聚集率%备注DEAE20150116峰6,0.665mg/ml-3.56絮团状物出现DEAE20150116峰6脱盐,0.473mg/ml-3.12DEAE20150116SP上样

10、液，0.502mg/ml-4.42SP流穿8-12t，0.175mg/ml0.28SP-31t0.157mg/ml0SP-28-32t0.153mg/ml32.71SP-34t0.198mg/ml3.09絮团状物出现SP-33-38t0.158mg/ml1.29SP-45t0.819mg/ml66.88SP-峰3,41-50t0.553mg/ml80.57SP-56t0.314mg/ml81.82SP-54-58t0.224mg/ml81.81时间：20150119层析柱规格16*20介质名称CM Bestarose FF层析介质量24ml样品名称DEAE (6HP)20150113 4-5峰

11、与5峰1：1混合（各30ml）上样量38.76mg蛋白样品处理用G-25柱交换HAC使 PH至4.5上样体积91ml程序如下：Time(min)BFlow(ml/min)Fracton(min/管）001.539001.536151.53220801.53BufferA：0.02mol/L PH 4.5 NaAC-HACBufferB：0.02mol/L PH 4.5 NaAC-HAC+1mol/L Nacl图谱如下：20150119，CM层析样品蛋白浓度10ug聚集率2ug聚集率20150119CM原样0.707mg/ml11.76絮团状物出过G-25后上样液0.406mg/ml75.53FT0.0.181mg/ml87.0644-45T0.226-7.13絮团状物出46-48T0.173-0.97絮团状物出49-50T（主峰1前沿）0.15347.7651-54T（主峰1）0.398-1.3973.5255-60T（主峰2）0.46751.3264-67T0.167对比空白71.88CM-3-46-48TCM-3-51-54TCM-3-55-60T主峰1CM-3-64-67T主峰2CM-3-原样Maker 对比CM-3-流穿CM-3-37-43TCM-3-44-45TCM-3-G25脱盐