Streptactin填料（Strep-tag蛋白纯化填料）

产品名称：Streptactin Agarose Resin 6FF for Strep-tag II Fusion Protein Purification， Streptactin填料（Strep-tag II蛋白纯化填料）

货号：P4136

品牌：Engibody

简介：Streptactin Resin, Strep-tag II蛋白纯化填料，用于Strep-tag II标签融合蛋白的纯化。

Streptactin Agarose Beads 6FF is a robust and stable affinity resin for purification of Strep II-tagged proteins

• High binding capacity yields high resolution, sharp concentrated peaks, and reproducible purifications

• Physiological conditions and mild elution preserves the activity of the target protein

• Compatible with a wide range of reducing agents, detergents, denaturants, and other additives

• Easily scaled up, and easily regenerated with 0.5M NaOH

• Convenient use with a peristaltic pump or chromatography system

Form

10 mL 50% slurry (5 mL settled beads); 20 mL 50% slurry (10 mL settled beads); 50 mL 50% slurry (25 mL settled beads);

Storage instruction

2 to 8°C, do not freeze

Beads Diameter

90 µm

Binding Capacity

Approx. 6mg Strep (II)-tagged protein/mL settled beads

Ligand

StrepTactin (∼5mg StrepTactin/mL settled beads)

Matrix

Agarose Beads 6FF beads, highly crosslinked 6% agarose

Maximum pressure

0.3 MPa, 3 bar

Chemical stability

stable in all commonly used buffers, 0.5M NaOH (regeneration and cleaning), reducing agents, denaturants, and detergents

Flow rate

<150 cm/hr

Storage buffer

1*PBS, containing 20% Ethanol

Protocols

Attention: Dynamic binding capacity (DBC) is defined as mg protein applied per mL chromatography medium at the point where the concentration of protein in the column effluent reaches a value of 10% of the concentration in the sample. DBC was tested here with GAPDH-Strep-tag II (Mr 37, 400). Binding capacity is protein dependent.

Buffer preparation

Binding/Wash buffer: 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8

Or PBS (20 mM sodium phosphate, 280 mM NaCl, 6 mM potassium chloride), pH 7.4

Elution buffer: 2.5 mM desthiobiotin in binding buffer

Regeneration buffer: 0.5 M NaOH

Or 1 mM HABA (2-[4’-hydroxy-benzeneazo] benzoic acid) in binding buffer

Purification Protocol

1. Column packing

2. Sample preparation

1). Adjust the sample to the composition of the binding buffer. For example, dilute the sample with binding buffer or buffer exchange using a desalting column

2). Pass the sample through a 0.22 μm or a 0.45 μm filter and/or centrifuge it immediately before applying it to the column. If the sample is too viscous, to prevent it from clogging the column dilute it with binding buffer, increase lysis treatment (sonication, homogenization), or add DNase/RNase to reduce the size of nucleic acid fragments.

3. Purification of Recombinant Strep II-tagged proteins

1). Remove the stoppers and connect the column to the system. Avoid introducing air into the column.

2). If the column has been stored in 20% ethanol, wash out the ethanol with at least 5 column volumes of distilled water or binding buffer at a flow velocity of 50 to 100 cm/h.

3). Equilibrate the column with at least 5 column volumes of binding buffer.

4). Apply the pretreated sample. A lower flow rate can be used during sample application to optimize performance.

5). Wash with 5 to 10 column volumes of binding buffer or until no material appears in the effluent.

6). Elute with 5 column volumes of elution buffer. The eluted fractions can be buffer exchanged using a prepacked desalting column

7). After elution, regenerate the column by following the procedure (see below).

4. After purification, the beads should be regenerated as follows:

1). Regenerate the column with 3 column volumes of distilled water followed by 3 column volumes of 0.5 M NaOH and 3 column volumes of distilled water.

2). Re-equilibrate the column with 5 column volumes of binding buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8 or PBS [20 mM sodium phosphate, 280 mM NaCl, 6 mM potassium chloride], pH 7.4), then store in the same volume of 1*PBS (containing 20% Ethanol) before starting the next purification.

An alternative to the above regeneration/re-equilibration is 15 column volumes of 1 mM HABA (2-[4’-hydroxy-benzeneazo] benzoic acid) in binding buffer followed by 30 column volumes of binding buffer. The displacement is detected by the change in color of the medium in the column from yellow to red. This color change is due to the accumulation of HABA/StrepTactin complexes. The HABA is washed away with the binding buffer.

Streptactin琼脂糖珠6FF是一种用于纯化Strep II标记蛋白的强健且稳定的亲和树脂

•高结合能力产生高分辨率、尖锐的集中峰和可重复的纯化

•生理条件和温和洗脱可保持目标蛋白的活性

•与多种还原剂、洗涤剂、变性剂和其他添加剂兼容

•易于放大，并易于用0.5M NaOH再生

•方便使用蠕动泵或色谱系统

类型

10 mL 50%浆料（5 mL沉淀珠子）；20 mL 50%浆料（10 mL沉淀珠子）；50 mL 50%浆料（25 mL沉淀珠子）；

存储说明

2至8°C，请勿结冰

beads直径

90µm

结合能力

约6mg Strep（II）标签融合蛋白/mL沉淀beads

配体

StrepTactin(∼5mg StrepTactin/mL沉淀beads）

基质

琼脂糖珠6FF珠，高交联6%琼脂糖

最大压力

0.3 MPa，3bar

化学稳定性

在所有常用缓冲液、0.5M NaOH（再生和清洗）、还原剂、变性剂和洗涤剂中稳定

流速

<150厘米/小时

存储缓冲液

1*PBS，含20%乙醇

注意：动态结合容量（DBC）定义为每mL色谱介质中蛋白质的mg蛋白质，此时柱液中的蛋白质浓度达到样品浓度的10%。DBC在这里用GAPDH Strep标签II（Mr 37400）进行了测试。结合能力是蛋白质依赖性的。

缓冲液制备

结合/洗涤缓冲液：100 mM Tris-HCl，150 mM NaCl，1 mM EDTA，pH 8

或PBS（20 mM磷酸钠、280 mM氯化钠、6 mM kcl]），pH 7.4

洗脱缓冲液：结合缓冲液中2.5 mM脱硫biotin

再生缓冲液：0.5 M NaOH

或结合缓冲液中的1mM HABA（2-[4'-羟基苯偶氮]苯甲酸）

纯化步骤

1.柱子里装上填料

2.样品制备

1). 将样品调整到绑定缓冲液的组成。例如，用结合缓冲液稀释样品或用脱盐柱交换缓冲液

2). 将样品通过0.22μm或0.45μm过滤器和/或离心，然后将其应用于色谱柱。如果样品太粘稠，为了防止其堵塞柱，用结合缓冲液稀释样品，增加裂解处理（超声处理、均质化），或添加DNase/RNase以减小核酸片段的大小。

3.重组Strep II标签融合蛋白的纯化

1). 拆下塞子并将立柱连接至系统。避免将空气引入塔中。

2). 如果柱已储存在20%乙醇中，则用至少5柱体积的蒸馏水或结合缓冲液以50至100 cm/h的流速冲洗乙醇。

3). 用至少5柱体积的结合缓冲液平衡柱。

4). 应用预处理过的样品。在样品应用过程中，可以使用较低的流速来优化性能。

5). 用5至10柱体积的结合缓冲液清洗，或直到流出物中没有物质。

6). 用5柱体积的洗脱缓冲液洗脱。洗脱的馏分可以使用预填充脱盐柱进行缓冲交换

7). 洗脱后，按照程序再生色谱柱（见下文）。

4.纯化后，珠子应按如下方式再生：

1). 用3柱体积的蒸馏水再生柱，然后用3柱容积的0.5M NaOH和3柱体积蒸馏水再生。

2). 用5柱体积的结合缓冲液（100 mM Tris HCl、150 mM NaCl、1 mM EDTA，pH 8或PBS[20 mM磷酸钠、280 mM NaCl、6 mM kcl]，pH 7.4）重新平衡色谱柱，然后在开始下一次纯化之前，将其储存在相同体积的1*PBS（含20%乙醇）中。

上述再生/再平衡的替代方案是在结合缓冲液中加入15柱体积的1mM HABA（2-[4’-羟基苯偶氮]苯甲酸），然后加入30柱体积的结合缓冲液。通过柱中介质的颜色从黄色变为红色来检测位移。这种颜色变化是由于HABA/StrepTactin复合物的积累。用结合缓冲液冲洗HABA。