文档介绍：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
第一页，共20页。
Column packing
Rabies vaccine
Sephar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
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第十页，共20页。
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 ( MPa) for XK columns.
3. If the packing equipment does not include a pressure 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 chromatographic procedures.
第十一页，共20页。
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 bar
3-5mm blow the settled bed was made
第十二页，共20页。
Column packing
Sepharcyl S HR
Flow rates given below. Pack the gel in STEP 1 for 2 hours or 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
第十三页，共20页。
Column packing
Superdex 75,200 pg
50% slurry
1 step: 20cm/h flow rate, 2 hour
2 step: step the pressure is held at 3 bar, 1hour
第十四页，共20页。
Column packing
Sephadex G-25
50% slurry
400 cm/h flow rate
3mm blow the settled bed was made
第十五页，共20页。
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。
第十六页，共20页。
在位清洗（CIP）
离子性吸附的蛋白: Wash with CV 2M NaCl. Contact time 15 min.
沉淀的、疏水性结合的蛋白及脂蛋白：1 M