AKTA--实验室层析柱装柱技术介绍ppt课件.ppt

1、实验室层析柱装柱技术介绍,1,Column packing,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,2,Column packing,Rabies vaccine Sepharose 4 Fast Flow 5% column vol sample,3,Column packing,每米塔板数：N=1600,每米塔板数：N=3500,4,Column packing,N/m= 5.54 (Ve/Vh)2100/L,As = b/a,5,Column packing,检测装柱效果一般用0.5% (30um介质)或1% (90um介质)

2、 柱体积的1%丙酮测柱效和峰形 The linear flow rate should be 30 cm/h for 34 m and 50 m media and 20 cm/h for 90 m media,6,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) x 60 线性流速 (cm/hr) = 揪揪揪揪揪揪 柱子横切面积 (cm2),Column packing,7,Column packing,It is important that the column is properly equilibrated be

3、fore the packing is evaluated ( 2 column volumes). It is preferable to run three test runs to see whether the test values are stable. If a poor result improves during this test, the reason can be that the column was not properly equilibrated. To check that the bed is stable, run the column at 70% pa

4、cking pressure for 20 hours and test it again (preferably three test runs). Note that pressure spikes may cause poor packing (cracking). If this happens, an air trap and a pressure relief valve are needed between the pump and the column. The pressure relief valve should be placed between the air tra

5、p and column.,8,Column packing,9,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,Column packing,10,用空柱子须留意.,使用调节器(flow adaptor) - 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(packing reservoir)，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

6、，如用SOURCE15介质装C柱和XK柱，须换上5mm的网,11,Column packing,Sepharose FF based gel: Sepharose Q, DEAE, SP, Phenyl 1. the 75 % slurry just before packing. 2. Ideally, Fast Flow media are packed at a con stant pressure not exceeding 3 bar (0.3 MPa) for XK columns. 3. If the packing equipment does not include a pr

7、essure gauge, use a packing flow rate of at least 400 cm/h (15 cm bed height, 25). If recommended pressure or flow rate can not be obrained, use the maximum flow the pump can deliver, this should also give a easonably well-packed gel. Do not exceed 75 % of the packing flow rate in subsequent chromat

8、ographic procedures.,12,Column packing,Sepharose 4 Fast Flow 50-75% slurry just before packing. 110 cm/h flow rate and the pressure should not be increased beyond 1.0 bar 3-5mm blow the settled bed was made,13,Column packing,Sepharcyl S HR Flow rates given below. Pack the gel in STEP 1 for 2 hours o

9、r until the gel has reached a constant height, then increase the flow rate to the value listed for STEP 2 and pack for 60 minutes.,Packing flow rates (ml/h): Column STEP 1 STEP 2 XK 16/70 60 110 XK 16/100 60 100 XK 26/70 150 300 XK 26/100 150 270 XK 50/100 500 800,14,Column packing,Superdex 75,200 p

10、g 50% slurry 1 step: 20cm/h flow rate, 2 hour 2 step: step the pressure is held at 3 bar, 1hour,15,Column packing,Sephadex G-25 50% slurry 400 cm/h flow rate 3mm blow the settled bed was made,16,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（1M NaCl)洗脱或改变pH。,17,在位清洗（CIP）,离子性吸附的蛋白: Wash with 0.5 CV 2M NaCl. Contact time 15 min. 沉淀的、疏水性结合的蛋白及脂蛋白：1 M NaCl at 40 cm/h, 1-2 hours. 脂类及非常强结合的疏水性蛋白：2-4 CV 0.5%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，2-4 CV 1-2 小时。,18,柱子的保存,0.15 mol/L NaAc pH 4.0 20% ethanol 0.01 mol/L NaOH 0.05% sodium