免疫亲和色谱柱制备方法

Instructions 71-7086-00 AF Affinity media

GE Healthcare CNBr-activated Sepharose? 4B CNBr-activated Sepharose 4B is a pre-activated medium for immobilization of ligands containing primary amines. It provides a very convenient way to immobilize ligands by the cyanogen bromide method. The coupling reaction is spontaneous, rapid and easy to carry out. The application area covers immobilization of proteins, peptides and nucleic acids.

通用电气医疗集团CNBr-activated琼脂糖?4 b CNBr-activated琼脂糖4 b是固定地激活介质的包含一级胺配体。它提供了一个非常方便的方法固定溴化氰配体的方法。偶联反应是自发的,快速和容易执行。应用领域涵盖固定的蛋白质、多肽和核酸。

Table 1. Medium characteristics.

Bead structure: 4% agarose

Spacer: None

Coupling capacity: 25–60 m g α-chymotrypsinogen/ml drained medium Bead size range: 45–165 μm

Average bead size: 90 μm

Max linear flow rate*: 75 cm/h at 25°C, HR 16/10 column, 5 cm bed height

pH stability**:

表1。媒介的特征。

琼脂糖珠结构:4%

间隔:没有

耦合能力:α-chymotrypsinogen外地毫克/毫升排水

媒介

珠大小范围:45 - 165μm

平均珠大小:90μm

最大线性流量*:75厘米/小时25°C,人力资源列16/10,5厘米床层高度pH值稳定* *:

Long term: 3–11

Short term: 2–11

Chemical stability***: Stable to all commonly used aqueous solutions. Can be used with non-ionic detergents, denaturing

solvents, e.g. 8 M urea and 6 M guanidine hydrochloride. Stable in organic solvents, such as 50% dimethylformamide and 50% dioxane. Autoclavable: Not recommended

长期:3-11

短期:2 - 11

化学稳定性* * *:所有常用的水稳定的解决方案。

可以用于非离子去污剂,变性

溶剂,例如8 M尿素和6 M胍盐酸。等有机溶剂、稳定50%二甲基甲酰胺和二氧六环的50%。

Autoclavable:不推荐

* Linear flw rate = volumetric flw rate (cm 3/h) /column cross-sectional

area (cm 2)

** The ranges given are estimates based on our knowledge and experience. Please note the following:

pH stability, long term refers to the pH interval where the gel is stable over a long period of time without adverse effects on its subsequent chromatographic performance.

*线性flw率=容积flw率(厘米3 /小时)

列横截面积(2厘米)

* *给出的范围的估计是基于我们的知识和经验。请

请注意以下几点:

pH值稳定、长期的pH值间隔指的是凝胶稳定在很长一段时间没有不利影响在其随后的色谱性能。

pH stability, short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.

*** Data refer to the coupled product, provided that the ligand can withstand the pH or chemical environment.

pH值稳定,短期内指的是再生的pH值间隔,原位清洗（CIP）和卫生处理程序。

* * *数据指的是耦合的产品,提供的配体能够承受或化学环境pH值。Contents

1. Preparing the medium 4

2. Coupling the ligand 4

3. Factors effecting the coupling efficiency 5

4. Packing Sepharose 4B 7

5. Binding 9

6. Elution 9

7. Regeneration 10

8. Storage 10

9. Further information 10

10. Ordering information 11

内容

1。准备中4

2。耦合配体4

3。因素影响耦合效率的5

4。包装琼脂糖4 b 7

5。绑定9

6。洗脱9

7。再生10

8。存储10

9。进一步的信息10

10。订购信息11

1. Preparing the medium

CNBr-activated Sepharose 4B is supplied lyophilized in the presence of additives. These additives must be washed away at low pH (pH 3) before

coupling the desired ligand. The use of low pH (pH 3) preserves the activity of the reactive groups, which otherwise hydrolyze at high PH. Weigh out the required amount of powder (1 g lyophilized powder gives about 3.5 ml final volume of medium) and suspend it in 1 mM HCl. The medium swells immediately and should now be washed for 15 minutes with1 mM HCl on a sintered glass filter (porosity G3). Use approximately 200 ml 1 mM HCl per gram freeze-dried powder, added in several aliquots. 1。

准备中

CNBr-activated琼脂糖4 b提供冻干的添加剂。这些添加剂必须冲走在低pH值(酸碱度3)耦合所需的配体。使用低pH值(酸碱度3)保存的活动活性组,否则水解在高Ph。称出所需的粉(1 g冻干粉给约3.5毫升最终介质体积)和盐酸暂停在1毫米。中立即膨胀,现在应该在烧结玻璃过滤器(孔隙度G3)用1毫摩/ml的盐酸清洗15分钟。使用约200毫升盐酸1毫米每克冷冻干燥粉,添加到数小份。

2. Coupling the ligand

General ligand coupling procedure

1. Dissolve the ligand to be coupled in coupling buffer, 0.1 M NaHCO3 pH 8.3 containing 0.5 M NaCl. Use about 5 ml coupling solution/g lyophilized powder. About 5–10 mg protein per ml medium is recommended. For smaller ligands add 1–10 μ moles per ml medium.

2. Add the coupling solution containing the ligand with the prepared

medium suspension in a stoppered vessel.

3. Rotate the mixture end-over-end for 1 h at room temperature or overnight at 4 °C. Other gentle stirring methods may be employed. Do not use a magnetic stirrers as these can disrupt the Sepharose beads.

2。耦合的配体

一般配位体耦合过程

1。配体溶解在耦合耦合的缓冲区,0.1 NaHCO3

pH值8.3包含0.5 M氯化钠。使用约5毫升耦合解决方案/ g冻干粉。建议5 - 10毫克每毫升的蛋白质中。小配体添加1 - 10每毫升μmoles 媒介。

2。添加包含配位体的耦合方案的准备中悬浮在一个密封的容器里。3。旋转end-overend混合物在室温下1 h或隔夜在4°C。其他温和的搅拌方法可以使用。不要使用磁力搅拌器可以扰乱琼脂糖珠。

4. Wash away excess ligand with at least 5 medium (gel) volumes of coupling buffer.

5. Block any remaining active groups. Transfer the medium to 0.1 M TrisHCl buffer, pH 8.0 or 1 M ethanolamine, pH 8.0. Let it stand for 2 hours.

6. Wash the medium with at least three cycles of alternating PH. Wash with at least 5 medium volumes of each buffer. Each cycle should consist of a wash with 0.1 M acetic acid/sodium acetate, pH 4.0. Containing 0.5 M NaCl followed by a wash with 0.1 M Tris-HCl, pH 8 containing 0.5 M

NaCl. 4。洗去多余的配位体至少有5倍(凝胶) 体积的缓冲液。

5。阻止任何剩余的活跃群体。媒介转移到0.1米TrisHCl缓冲区,pH 值8.0或1米乙醇胺,pH值8.0。让它站了2小时。

6。洗中至少有三个周期交替ph洗至少有5中每个缓冲区的容量。每个周期都应该由一个洗0.1乙酸/醋酸钠,pH值4.0包含0.5 M氯化钠与0.1 Tris-HCl接着洗,pH值8包含0.5 M氯化钠。

3. Factors effecting the coupling efficiency（3影响耦合率因素）

pH

The coupling reaction proceeds most efficiently in the pH range 8–10 where the amino groups on the ligand are predominantly in the unprotonated form. A buffer at pH 8.3 is most frequently used for coupling proteins. Coupling at low pH is less efficient but may be advantageous if the ligandlooses biological activity when it is fixed firmly by multi-point attach mentor if steric hindrance between binding sites occurs when a large amount of high molecular weight ligand is immobilized. A buffer of approximately pH 6 is used for coupling at low pH.

PH值

偶联反应所得最有效的pH值范围内8 - 10

配体上的氨基基团主要是来自于未质子化

的形式。缓冲区在pH值8.3是最常用于耦合蛋白质。

耦合在低pH值效率较低,但可能advantegeous如果配体

失去生物活性,通过多点附件固定牢固

或者如果位阻之间的绑定时发生大量的网站

高分子量配位体是固定的。缓冲约pH值6用于耦合在低pH值。Coupling solution

Coupling should be performed in bicarbonate or borate buffers. Tris and other buffer salts containing amino groups should not be used since these will couple to the medium.

Organic solvents may be needed to dissolve the ligand. Dimethylformamide and dioxane may be used up to 50% of the final mixture. The same concentration of organic solvents should be included in the coupling buffer. Always adjust the pH after dissolving the ligand, since organic solvent usually lowers pH.

耦合的解决方案

耦合应该执行重碳酸盐或硼酸缓冲。三羟甲基氨基甲烷和液

其他缓冲盐含有氨基酸群体不应该因为这些使用将两介质。

可能需要有机溶剂来溶解配体。二甲基甲酰胺和二氧六环可以使用多达50%的最终的混合物。相同浓度的有机溶剂应包括在耦合缓冲区。总是在溶解后调整pH值配位体,因为有机溶剂通常降低pH值。

Salt

To minimize protein-protein adsorption and the formation of protein aggregates, it is recommended to have a high salt content, 0.5 M NaCl, in the coupling buffer.

Temperature

Coupling is completed within 2 hours at room temperature, 20–25 °C. If cold room temperatures are necessary, coupling can be carried out overnight.

盐

减少蛋白质吸附和蛋白质的形成总量,建议盐含量高,0.5 M氯化钠,在耦合缓冲区。

温度

耦合是在室温下2小时内完成,20 - 25°C。如果冷房间的温度是必要的,可以在一夜之间进行耦合。

Ligand concentration

A very high ligand concentration can have adverse effects on affinity chromatography. Firstly, the binding efficiency of the adsorbent may be reduced due to steric hindrance between the active sites. Secondly, substances are more strongly bound to the immobilized ligand and this may result in difficult elution. Thirdly, the extent of non-specific binding increases at high ligand concentrations. For an efficient adsorbent, 1–10 μ moles ligand per ml medium is recommended. For protein ligands, 5–10 mg protein per ml medium is recommended.

配位体浓度

非常高的配位体浓度会有负面影响亲和色谱法。首先,绑定吸附剂的效率可能会减少由于位阻之间的活跃的网站。其次,物质更强烈地绑

定到固定化的配位体,这可能导致难洗脱。第三,非特异性结合的程度在高配位体浓度增加。对于一个高效的吸附剂,1 - 10μmoles每毫升配体中推荐。对蛋白质配体5 - 10毫克每毫升蛋白质中推荐。Controlling the coupling effiiency

Sometimes it may be necessary to reduce the number of coupling groups on the matrix to preserve the structure of the binding site in a labile molecule, or to facilitate elution when high binding constants make elution difficult or when steric effects reduce the binding efficiency of a large ligand. Reduced coupling activity may be achieved by controlled hydrolysis of the activated medium prior to coupling, or by coupling at a lower pH. Prehydrolysis reduces the number of active groups available for coupling and reduces the number of points of attachment between the protein and matrix as well as the amount of protein coupled. In this way a higher binding activity of the product is obtained. At pH 3, coupling activity is lost only slowly, whereas at pH 8.3 activity is lost fairly rapidly.

A large molecule is coupled at only about half as many points after 4 h pre-hydrolysis at pH 8.3 as it is before the number of active groups was reduced.

控制耦合effiiency

有时可能需要减少耦合组矩阵保存结合位点的结构不稳定分子,或促进洗脱时高绑定常量使洗脱困难或立体效果降低的绑定效率大的配体。减少耦合活动可以通过控制水解前激活介质的耦合,或通过耦合

在一个较低的博士Prehydrolysis减少可用于耦合活动组的数量,减少点数量的蛋白质之间的依恋和矩阵以及蛋白质的耦合。这样一个更高的绑定产品的活动。在pH值3,耦合活动缓慢,而在pH值8.3活动失去了相当迅速。大量分子耦合后只有大约一半的点4 h pre-hydrolysis pH值8.3,因为它是在活动组织的数量减少。

Blocking excess remaining groups

Remaining active groups on the medium should be deactivated or blocked after the coupling. These can be hydrolyzed in a mildly alkaline pH (2 hours at room temperature or 16 h at 4 °C). Alternatively, these can also be blocked by adding an excess of a small primary amine (e.g. Tris-HCl, ethanolamine, glycine) at approximately pH 8 (2 hours at room temperature or 16 h at 4 °C). These blocking agents introduce a small number of charged groups into the medium. The effect of these charged groups is overcome by the use of a relatively high salt concentration (0.5 M NaCl) in the buffer for affinity chromatography.

阻塞超额剩余组

保持活跃的团体中耦合后应停用或阻塞。这些可以水解中和碱性pH 值(在室温下2小时或16小时在4°C)。另外,这些也可以被添加过多的一个小伯胺(例如Tris-HCl、乙醇胺、甘氨酸)大约在pH值8(2小时在室温下或16 h在4°C)。这些阻塞代理中引入少量的带电组中。这些带电组的影响通过使用一个相对较高的盐浓度(0.5 M氯化钠)在缓冲亲和色谱法来克服。

Washing the adsorbent

To remove excess of uncoupled ligand after coupling, the adsorbent is washed alternatively with high and low pH buffer solutions at least three times. Acetate buffer (0.1 M, pH 4) and coupling buffer (pH 8.3) each containing 0.5 M NaCl are suitable. This procedure ensures that no free ligand remains ionically bound to the immobilized ligand.

洗吸附剂

去除多余的非耦合配位体耦合后,吸附剂洗或者高和低pH缓冲方案至少三次。醋酸缓冲(pH值0.1米,4)和耦合缓冲区(pH值8.3)每个包含0.5 M氯化钠是合适的。这个过程可以确保没有多余的配位体仍然以离子键绑定到固定化的配体

4. Packing Sepharose 4B

Prepare a slurry with binding buffer, see below, in a ratio of 75% settled medium to 25% buffer. The binding buffer should not contain agents which significantly increase the viscosity. The column may be equilibrated with viscous buffers at reduced flow rates after packing is completed.

1. Equilibrate all material to the temperature at which the chromatography will be performed.

2. De-gas the medium slurry.

3. Eliminate air from the column dead spaces by flushing the end pieces with buffer. Make sure no air has been trapped under the column net.

Close the column outlet with a few centimeters of buffer remaining in the column.

4。包装琼脂糖4 b

准备一个泥浆与绑定缓冲,见下文,比率为75%解决介质缓冲的25%。绑定缓冲不应该包含药物，会大大增加粘度。列在流量减少可能与粘性平衡缓冲包装完成后。

1。平衡所有材料的温度色谱法将被执行。

2。悬浮液排气。

3。消除空气冲洗结束后从列死亡空间碎片与缓冲区。确保没有空气困在列。近几厘米的列出口缓冲剩余的列

4. Pour the slurry into the column in one continuous motion. Pouring the slurry down a glass rod held against the wall of the column will minimize the introduction of air bubbles.

5. Immediately fill the remainder of the column with buffer, mount the column top piece onto the column and connect the column to a pump.

6. Open the bottom outlet of the column and set the pump to run at the desired flow rate. This should be at least 133% of the flow rate to be used during subsequent chromatographic procedures. However, the maximum flow rate, see Table 1, is typically employed during packing.

Note: If you have packed at the maximum linear flow rate, do not exceed 75% of this in subsequent chromatographic procedures.

7. Maintain the packing flow rate for 3 bed volumes after a constant bed

height is reached.

4。将浆倒入列在一个连续的运动。浇注泥浆下举行的玻璃棒靠墙的列将减少气泡的引入。

5。立即用缓冲填充剩余的柱,柱山顶块到列和列连接到泵。

6。打开底部出口的列和泵运行所需的流量。这应该是至少133%的流量在随后的色谱过程中使用。然而,最大流量,见表1,通常使用在包装。注意:如果您有包装最大线性流量,不超过75%的在随后的色谱过程。7。维护包装流量3倍柱体积

Using an adapter

Adapters should be fitted as follows:

1. After the medium has been packed as described above, close the column outlet and remove the top piece from the column. Carefully fill the rest of the column with buffer to form an upward meniscus at the top.

2. Insert the adapter at an angle into the column, ensuring that no air is trapped under the net.

3. Make all tubing connections at this stage. There must be a bubble-free liquid connection between the column and the pump.

4. Slide the plunger slowly down the column so that the air above the net and in the capillary tubings is displaced by eluent. Valves on the inlet side of the column should be turned in all directions during this procedure to ensure that air is removed.

使用一个适配器

适配器应当安装如下:

1。中包装后如上所述,关闭列出口和删除顶部的列。仔细填写其余的列与缓冲顶端形成一个向上的半月板。

2。适配器在一个角度插入列,确保没有空气困在网。

3。让所有油管连接在这个阶段。必须有一个含气泡液体柱和泵之间的联系。

4。慢慢滑柱塞的列,上面的空气净和洗脱液的毛细管套管是流离失所。阀门的进口侧列应该把四面八方在此过程以确保空气被移除。

5. Lock the adapter in position on the medium surface, open the column outlet and start the eluent flow. Pass eluent through the column at the packing flow rate until the medium bed is stable. Re-position the adapter on the medium surface as necessary. The column is now packed and equilibrated and ready for use.

5。锁适配器在介质表面的位置,打开列插座,启动洗脱液流。洗脱液通过列在包装流量,直到介质床上是稳定的。在必要时重新放置介质表面上的适配器。柱子现在已经包装和平衡,并且可以使用了

5. Binding

Conditions for binding depend on which ligand is used. Literature references and textbooks may give good guidelines. The adsorption will depend upon parameters such as sample concentration, flow rate, pH, buffer composition and temperature. General guidelines for adsorption are:

? Sample pH should be th e same as that of the binding buffer. Filter the sample through a 0.22 μm or 0.45 μm filter to prolong the working life of the medium.

? After the sample has been loaded, wash the medium with binding buffer until the base line is stable.

5。绑定

使用绑定条件依赖于配体。文献引用和教科书可能提供良好的指导方针。吸附等参数取决于样品浓度、流量、pH值、缓冲区组成和温度。吸附的一般规则是:

?样本pH值应该一样绑定缓冲。筛选样本通过一个0.22μm或0.45μm 过滤器延长工作寿命的媒介。

?样本被加载后,清洗介质与绑定缓冲直到基线稳定

6. Elution

Conditions for elution of bound substances depend on which ligand is used. Literature references and textbooks may give good guidelines. General guidelines are described below.

? pH change: A change in pH alters the degree of ionization of charged groups at the binding sites. Elution is generally affected by a decrease in pH. The chemical stability of the matrix, ligand and adsorbed substances determines the limits of pH which may be used.

? Ionic strength: A buffer with increased ionic strength is used. Elution with a continuous or step-wise gradient may be used. A gradient of

increasing salt concentration can be used to separate substances bound to the adsorbent. NaCl is most frequently used and enzymes usually elute at a concentration of 1 M NaCl or less. If the interaction has a very high affinity, a chaotropic salt may be required.

6。洗脱

洗脱条件束缚物质的使用依赖于配体。文献引用和教科书可能提供良好的指导方针。一般的指导方针如下所述。

?pH值变化:pH值的变化改变了电离度的带电组结合位点。洗脱普遍下降的影响博士矩阵的化学稳定性,配体和吸附物质决定了pH值的限制可能被使用。

?离子强度:使用缓冲区与离子强度增加。与连续或步进式梯度洗脱可以使用。增加盐浓度的梯度可以用来分离物质绑定到吸附剂。氯化钠是最常用的浓度和酶通常洗提1 M氯化钠或更少。如果交互有很高的亲和力,可能需要一个离液序列高的盐。

? Competitive elution: Competitive eluents are often used to selectively elute substances from a group specific adsorbent and also when the affinities are relatively low. Selectively retained substances are usually displaced at low concentrations of eluting agents, often less then 10 mM. Either continuous or step-wise gradients may be used.

? Reduced polarity: Conditions which lower the polarity of the eluent to promote elution may be used if they do not inactivate eluted substances. Dioxane (up to 10%) or ethylene glycol (up to 50%) may be used.

? Deforming eluents: If the elution methods described above fail to affect elution, deforming agents, such as chaotropic salts, guanidine-HCl or urea, which alter the structure of the proteins can be used.

?竞争洗脱:竞争洗脱液通常用于有选择地洗提物质从一组特定的吸附剂和当亲和力也相对较低。有选择地保留物质通常是流离失所的洗脱剂的低浓度,通常不到10毫米。可以使用连续或步进式梯度。

?减少极性:条件较低的极性洗脱液促进洗脱可以使用如果他们不灭活洗提物质。二氧六环(10%)和乙二醇(50%)可能被使用。

?变形洗脱液:如果上述洗脱方法不影响洗脱,变形,如离液序列高的盐,盐酸胍或尿素,改变蛋白质的结构都可以使用。

7. Regeneration

Conditions for regeneration depend on which ligand has been coupled. Literature references and textbooks may give good guideines. A general regeneration method is described below:

An affinity medium may be regenerated for re-use by washing the medium with 2–3 column volumes of alternating high pH (0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5) and low pH (0.1 M sodium acetate, 0.5 M NaCl, pH 4.5) buffers. This cycle should be repeated 3 times followed by re-equilibration in binding buffer.

7。再生

再生条件依赖于配体耦合。文献引用和教科书guideines可能给好处。一个通用的再生方法描述如下:

一个亲和介质可再生重复使用的洗涤2 - 3柱体积的介质交替高pH值(0.1 M Tris-HCl,0.5 M氯化钠,pH值8.5)和低pH值(0.1 M醋酸钠,0.5 M 氯化钠,pH值4.5)缓冲区。这个周期应该重复3次在绑定缓冲重平衡紧随其后。

8. Storage

Lyophilized CNBr-activated Sepharose 4B should be stored below 8 °C. Swollen coupled medium should be stored at 4–8 °C in presence of a bacteriostatic agent, e.g. 20% ethanol.

8。存储

冻干CNBr-activated琼脂糖4 b应该存储8°C以下。肿胀的耦合介质应该存储在4 - 8°C的抑菌剂,如20%的乙醇。