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离子交换层析

子交换介质
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Troubleshooting
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Screening for the optimal ion exchanger
Sample:
Columns:
Start buffer: Elution buffer: Flow rate: Running parameters:
5
Lane 1: Lane 2: LMW markers Lane 3: Starting material Lane 4: Flow through Lane 5: 1st peak
(containing α-amylase) Lane 6: 2nd peak Lane 7: LMW markers
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Monodisperse media
MiniBeads™ 3 µm micro-purification MonoBeads™ 10 µm polishing SOURCE™ 15 15 µm polishing SOURCE™ 30 30 µm intermediate steps
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EDTA absorbs at 254 nm
RNA/DNA
%B
100
RNA
DNA
50
0
0
2
4
6
8
Time (min)
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Elution schemes
• Isocratic • Step gradient • Linear gradient • Adapted
– Not usual(浓缩) – Excellent for capture steps – Standard method – Saves time and improves results
• pH gradient • Salt gradient • Both pH and salt
– Seldom used – Standard method – Can be tricky!
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Gradient vs step elution
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浓缩
STREAMLINE DEAE: rec α-amylase, E. coli
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多功能: DNA 结合蛋白的纯化
Technique CIEX AC AC AC
HeLa cell nuclear extracts
SP Sepharose High Performance
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起始缓冲液pH的选择
1) 1 ml 介质放入试管 2) 加不同的 pH‘s缓冲液 3) 加样品混合 4) 分析上清液中蛋白的称分
Here the target binds completely at pH 7.5 and above.
Conclusion:
Anion exchanger, initial pH 7.5.
缓冲液的使用
• 20–50 mM 缓冲液
• 确定正确的 pH 值, 溶解样品,
以及洗脱缓冲液
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pH value and buffer capacity
pH value
AU
AU
pH value
gradient
gradient
volume
• 正确的pH值样品
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• Final polishing, 20 µg protein obtained
多功能: 膜蛋白的纯化
Technique
Placenta extract in 1.5% Triton X-100
AC
Blue Sepharose
AIEX
DEAE Sephacel
CIEX
SP Sepharose FF
COOR NH+3
缓冲液pH的选择
14
10 pH
COO-
R NH2
利用 pH控制分离效果
pH is an excellent parameter by which to control selectivity in IEX
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选择正确的离子交换介质
• 没有一种离子交换是非常完美的 ! • 不同的样品、不同的纯化目的需要不同的离
• 适用于所有的纯化阶段及所有的规模生产 • 可控制的 • 高选择 • 高载量 • 可以浓缩样品 • 高回收率
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捕获阶段
• 在纯化的初始阶段从上清中直接捕获生物分子
Q Sepharose XL: rec α-amylase, E. coli
123 4 5 6 7
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可调控的纯化
结果可以选择多种因子优化
Effect of pH on fractionation of model proteins
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小规模纯化
Starting material Peak 2
Mini Q PC 3.2/3: Glutathione synthetase
5
• Rapid capture
8
9 2447 4943
7
• General AC step for DNA binding proteins
• Removal step, non-specific DNA binding activity removed
• Main purification step
HiTrap IEX Selection Kit: conalbumin (I), α-lactoglobulin (II) and soya bean trypsin inhibitor (III)
介质的优化
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Matrix and speed
X
Mechanically strong beads work at high flow rates
离子交换层析原理
适用于所有的纯化阶段和所有的 放大生产
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内容
• 一些典型应用 • 原理 • 实际操作方法
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什么是离子交换层析?
离子交换层析法是一种吸附层析, 是以分子的电荷差异来分离的。
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为什么使用离子交换层析法?
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SDS-PAGE
Packing a 2m CHROMAFLOW™ column with Q Sepharose™ Big Beads
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离子交换原理
操作的步骤
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平洗再平上衡脱样衡生12
通过电荷的差异分离
6.0
6.5
7.0
7.5
8.0
pH
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Determining optimal pH by scouting
pH scouting for the separation of pancreatin
ÄKTA Scouting
mAU
50
40
30
20
10
0
0
20
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+
Overall charge on protein
acid
excess positive charge
isoelectric point
alkaline
balanced positive and negative charge excess negative charge
COOH
R NH+3
3
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volume
正确的pH值洗脱缓冲液
pH 0.5
pH 0.5
A: Citrate 20 mM, pH 4.9
B: Citrate 20 mM, NaCl 1 M, pH 4.9
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A: Citrate 20 mM, pH 4.9 B: A + NaCl 1 M
EDTA在阴离子交换中的影响
4,5
4.5 • High sample throughput • High productivity
9 Lower the flow rate for:
9 • Maximum resolution
0 0
0
0,03
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0,02
200 cm/h (0.55 ml/min) 0,01
0
0
40
0
Time (min)
Heparin Sepharose Fast Flow
DNA-1 Sepharose
DNA-2 Sepharose
CIEX
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Mono S
Purification factor
Comment
J. Berthelsen et al. (1996) J. Biol. Chem. 271, 3822-3830
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Optimising flow rate
0,03
1800 cm/h
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