链霉亲和素磁珠

产品名称：Streptavidin Magnetic Beads，Streptavidin磁珠

货号： IF9042

品牌：Engibody

简介：Streptavidin磁珠，链霉亲和素磁珠是许多应用的理想选择，包括蛋白质和核酸的纯化、蛋白质相互作用研究、免疫沉淀、免疫分析、噬菌体展示、生物筛选、药物筛选和细胞分离。

Streptavidin Magnetic Beads are ideal for numerous applications, including purification of proteins and nucleic acids, protein interaction studies, immunoprecipitation, immunoassays, phage display, biopanning, drug screening and cell isolation.

Add the beads to a sample containing biotinylated molecules, e.g. peptides, proteins, antibodies, oligonucleotides, DNA/RNA. During a short incubation, the biotinylated molecule will bind to the beads. Separate the molecule-bead capture, washing, and detection can be optimized for manual or automated use. With indirect capture, mix the biotinylated molecule with the sample to capture the molecule target complex before adding the beads. Indirect target capture is an advantage when molecule-target kinetics are slow, affinity is weak, molecule concentration is low, or molecule-target binding requires optimal molecule orientation and true liquid-phase kinetics.

Streptavidin is covalently coupled to a magnetic particle. Streptavidin Magnetic Beads can be separated from solution using our 6-Tube Magnetic Separation Rack or 12-Tube Magnetic Separation Rack which concentrates the beads to the side of the tube instead of the bottom. This eliminates centrifugation steps, minimizes sample loss, increases washing efficiency, and saves time.

Tested applications

For coupling of proteins, antibodies, peptides and other molecules. Optimal amount should be determined by the end user.

Specificity

Binding to biotinylated molecules, e.g. nucleic acids, peptides, proteins, antibodies.

Form

Beads in Suspension (10 mg/mL), supplied in phosphate buffered saline (PBS) pH 7.4, with 0.1% bovine serum albumin (BSA) and 0.02% sodium azide as preservatives.

Storage instruction

Store at 4°C. This product is stable for 12 months. Do not freeze, dry or centrifuge beads. This may cause irreversible aggregation and decreased binding capacity.

Beads Diameter

1 µm

Binding Capacity

>100nmol Free biotin/mL beads slurry

200μg Biotinylated antibody/mL beads slurry

链霉亲和素磁珠是许多应用的理想选择，包括蛋白质和核酸的纯化、蛋白质相互作用研究、免疫沉淀、免疫分析、噬菌体展示、生物筛选、药物筛选和细胞分离。

将珠子添加到含有biotin化分子的样品中，例如肽、蛋白质、抗体、寡核苷酸、DNA/RNA。在短时间孵育期间，biotin化分子将与珠子结合。分离分子珠捕获、洗涤和检测可以优化为手动或自动使用。通过间接捕获，在添加珠子之前，将biotin化分子与样品混合以捕获分子-靶复合物。当分子靶动力学慢、亲和力弱、分子浓度低时，间接靶捕获是一个优势

低或分子靶结合需要最佳的分子取向和真实的液相动力学。

链霉亲和素与磁性粒子共价偶联。链霉亲和素磁珠可以使用我们的6管磁选架或12管磁选机架从溶液中分离，该机架将磁珠集中在管的侧面而不是底部。这消除了离心步骤，最大限度地减少了样品损失，提高了洗涤效率，节省了时间。

经过测试的应用

用于蛋白质、抗体、肽和其他biotin标记分子的偶联。最佳用量应由最终用户确定。

特异性

与biotin化分子偶联，如核酸、肽、蛋白质、抗体。

类型

悬浮珠（10 mg/mL），在pH 7.4的磷酸盐缓冲盐水（PBS）中提供，0.1%牛血清白蛋白（BSA）和0.02%防腐剂。

存储说明

储存于4°C。该产品稳定12个月。请勿冷冻、干燥或离心珠粒。这可能导致不可逆聚集和结合能力降低。

磁珠直径

1µm

结合偶联能力

>100nmol游离biotin/mL珠浆

200μg的biotin化抗体/mL珠浆

Protocols

Additional information

1, Streptavidin Magnetic Beads should be resuspended well before used.

2, Beads cannot be reused.

Procedure for binding biotinylated target directly.

A. Preparation of Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. Recommended coupling/washing buffers

protein/antibody samples: PBS buffer, pH7.4, nucleic acids samples: TES buffer

2. Elution buffer (only for biotinylated target purification): 8M Guanidine hydrochloride, pH1.5

B. Wash Streptavidin magnetic beads

Calculate the amount of beads required based on their binding capacity (see the above Binding Capacity),  and transfer the beads to a new tube.

1. Resuspend the beads in the vial (i.e. vortex for >30 sec, or tilt and rotate for 5 min).

2. Transfer the desired volume of beads to a tube.

3. Add an equal volume of Washing buffer, or at least 1 mL, and mix (vortex for 5 sec, or keep on a roller for at least 5 min).

4. Place the tube on a magnet for 1 min and discard the supernatant.

5. Remove the tube from the magnet and resuspend the washed beads in the same volume of washing buffer as the initial volume of beads taken from the vial (Step 2).

C. Binding proteins

1. Incubate the beads and biotinylated proteins in PBS buffer (pH 7.4) for 30 min at room temperature using gentle rotation.

2. Separate the coated beads with a magnet for 2–3 min.

3. Wash the coated beads 4–5 times in PBS containing 0.1% BSA.

4. Resuspend to the desired concentration for your application.

NOTE:

Elution of the biotinylated proteins that are bound to the Streptavidin Beads requires harsh conditions, such as boiling for 5 minutes with 8M guanidine•HCl, pH 1.5. Protein may leach from the beads with such conditions after centrifugation.

Procedure for Immunoprecipitation Using a Biotinylated Antibody (Binding Antigen Indirectly).

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. Recommended coupling/washing buffers

Phosphate-buffered saline (PBS) consisting of 100mM sodium phosphate, 150mM NaCl, pH 7.2.

2. Elution buffer (only for antigen purification by binding biotinylated antibody)

Low pH elution buffers such as 0.1M glycine•HCl, pH 2.5-2.8 are effective for most antibody-antigen interactions. However, the low pH condition may cause streptavidin to leach from the beads, resulting in a less pure immunoprecipitation product and preventing reuse of the beads.

3. Neutralization buffer: 1 M Tris•HCl, pH 8.5

4. Biotinylated antibody: 5-100µg, diluted in 50-1000µL of PBS buffer. Use an amount that can be easily bound by the amount of beads used (e.g.,<100µg antibody per mL of Streptavidin Beads Solution)

5. Antigen-containing Sample: 50-1500µL in a buffer that is compatible with antibody binding.

B. Procedure

1. Shake the bottle of Streptavidin Beads to resuspend the beads; then pipette 0.25-1mL into a microcentrifuge tube.

2. Place the tube in the Magnet to separate the beads; when the supernatant becomes clear, gently aspirate the supernatant and discard it.

3. Add 1mL of Binding/Wash Buffer, remove tube from magnet, and invert the tube several times to resuspend the beads. Then magnetically separate the beads, and remove and discard the supernatant.

4. Repeat Step 3 two additional times for a total of three washes.

5. Add 5-100µg of Biotinylated Antibody, remove tube from magnet, and gently invert tube several times to mix.    Incubate at room temperature for 30 minutes with constant or periodic mixing.

6. Place the tube in the Magnet to separate the beads; remove the supernatant, which contains any antibody that did not bind to the beads.

7. Add 0.1-1.5mL Antigen Sample, remove tube from magnet, and gently invert tube several times to mix. Incubate at room temperature for 30 minutes with constant or periodic mixing.

8. Magnetically separate the beads and remove the supernatant, which contains nonbound components of the sample.

9. Add 1mL of Binding/Wash Buffer, remove tube from magnet, and invert the tube several times to resuspend the beads. Then magnetically separate the beads and remove and discard the supernatant.

10. Repeat Step 9 two additional times for a total of three washes. If the Gentle Ab/Ag Elution Buffer will be used for elution, then use a non-phosphate buffer for at least the last one of these wash steps.

11. Add a small volume (typically 30-50μL) of Elution Buffer(0.1M glycine•HCl, pH 2.5-2.8), remove tube from magnet, and invert or gently vortex the tube several times to resuspend the beads in the buffer. Then magnetically separate the beads and remove the solution, which is an elution fraction containing the immunoprecipitated antigen.

12. Repeat Step 11 two additional times for a total of three elution fractions. Then add 10μL Neutralization buffer for 100μL elution fraction.

13. Analyze the elution fractions. If a high salt elution buffer was used, dialyze or desalt the sample before attempting to mix with loading buffer for SDS-PAGE.

General guidelines

• Keep the tube on the magnet for 2 min to ensure that all the beads are collected on the tube wall.

• For diluted samples, increase the incubation time or isolate in smaller batches using the same beads in each batch.

• Avoid air bubbles during pipetting.

• Free biotin in the sample will reduce the binding capacity of the beads. A disposable separation column or a spin column will remove unincorporated biotin.

• For some applications it can be an advantage to add a detergent such as 0.01–0.1% Tween™ 20 to the washing/binding buffers to reduce non-specific binding.

操作流程

Attetion

1、链霉亲和素磁珠在使用前应重新悬浮。

2、珠子不能重复使用。

直接结合biotin化靶的程步骤。

A、 溶液和试剂的制备

注：用反渗透去离子水（RODI）或同等级别的水制备溶液。

1.推荐的coupling/washing buffers

蛋白质/抗体样品：PBS缓冲液，pH7.4，核酸样品：TES缓冲液

2.洗脱缓冲液（仅用于biotin化目标纯化）：8M盐酸胍，pH1.5

B、 清洗链霉亲和素磁珠

根据珠子的结合能力计算所需的珠子数量（参见上述结合能力），并将珠子转移到新的试管中。

1.将珠子重新悬浮在小瓶中（即涡流>30秒，或倾斜并旋转5分钟）。

2.将所需体积的珠子转移到试管中。

3.加入等量的洗涤缓冲液，或至少1mL，并混合（涡旋5秒，或在滚筒上保持至少5分钟）。

4.将试管置于磁铁上1分钟，并丢弃上清液。

5.从磁铁上取下管，将洗涤过的珠粒重新悬浮在与从小瓶中取出的珠粒初始体积相同体积的洗涤缓冲液中（步骤2）。

C、 结合蛋白

1.将珠粒和biotin化蛋白在PBS缓冲液（pH 7.4）中在室温下使用温和旋转孵育30分钟。

2.用磁铁分离涂有涂层的珠子2-3分钟。

3.在含有0.1%BSA的PBS中清洗涂覆的珠粒4-5次。

4.重新悬浮至您所需的浓度。

注意：

与链霉亲和素珠结合的biotin化蛋白质的洗脱需要苛刻的条件，例如用8M胍•HCl，pH 1.5煮沸5分钟。离心后，蛋白质可能在这种条件下从珠中浸出。

使用biotin化抗体（间接结合抗原）进行免疫沉淀的程序。

A、 溶液和试剂

注：用反渗透去离子水（RODI）或同等级别的水制备溶液。

1.推荐的coupling/washing buffers

磷酸盐缓冲盐水（PBS），由100mM磷酸钠、150mM NaCl、pH 7.2组成。

2.洗脱缓冲液（仅用于通过结合biotin化抗体进行抗原纯化）

低pH洗脱缓冲液，如0.1M的glycine•HCl，pH 2.5-2.8，对大多数抗体-抗原相互作用有效。然而，低pH条件可能会导致链霉亲和素从珠子中浸出，导致较低纯度的免疫沉淀产物，并防止珠子的重复使用。

3.中和缓冲液：1 M Tris•HCl，pH 8.5

4.biotin化抗体：5-100µg，稀释在50-1000µL PBS缓冲液中。使用的量应易于与所用珠粒的量结合（例如，每毫升链霉亲和素珠粒溶液中的抗体<100µg）

5.含抗原样品：在与抗体结合相容的缓冲液中，50-1500µL。

B、 步骤

1.摇动链霉亲和素珠瓶，使珠重新悬浮；然后将0.25-1mL移液管移入微量离心管中。

2.将管放置在磁铁中以分离珠子；当上清液变得清澈时，轻轻抽吸上清液并丢弃。

3.加入1mL的binding/wash缓冲液，从磁铁上取下试管，并将试管倒置几次以重新悬浮珠子。然后用磁力分离珠子，取出并丢弃上清液。

4.再重复步骤3两次，总共清洗三次。

5.加入5-100µg的biotin化抗体，从磁铁上取下试管，轻轻翻转试管数次混合。在室温下孵育30分钟，持续或定期混合。

6.将管放置在磁铁中以分离珠子；去除上清液，上清液中含有任何不与珠子结合的抗体。

7.加入0.1-1.5mL抗原样品，从磁铁上取下试管，轻轻翻转试管数次以混合。在室温下孵育30分钟，持续或定期混合。

8.磁性分离珠子并去除上清液，上清液含有样品的非结合成分。

9.添加1mL的binding/wash缓冲液，从磁铁上取下试管，并将试管倒置几次以重新悬浮珠子。然后用磁力分离珠子，取出并丢弃上清液。

10.再重复步骤9两次，总共清洗三次。如果将使用温和的Ab/Ag洗脱缓冲液进行洗脱，则至少在最后一个洗涤步骤中使用非磷酸盐缓冲液。

11.加入少量（通常为30-50μL）洗脱缓冲液（0.1M glycine•盐酸，pH 2.5-2.8），从磁铁上取下试管，翻转或轻轻旋转试管几次，使珠粒重新悬浮在缓冲液中。然后磁性分离珠子并除去溶液，该溶液是含有免疫沉淀抗原的洗脱部分。

12.对总共三个洗脱组分再重复步骤11两次。然后添加10μL中和缓冲液，用于100μL洗脱组分。

13.分析洗脱组分。如果使用高盐洗脱缓冲液，在尝试与SDS-PAGE loading缓冲液混合之前，透析或脱盐样品。

注意事项

•将管保持在磁铁上2分钟，以确保所有珠子都收集在管壁上。

•对于稀释的样品，增加培养时间或在每批中使用相同的珠粒进行小批量分离。

•移液过程中避免气泡。

•样品中的游离biotin会降低珠子的结合能力。一次性分离柱或旋转柱将去除未结合的biotin。

•对于某些应用，添加洗涤剂（如0.01–0.1%吐温）是一种tween™ 20与洗涤/结合缓冲液结合以减少非特异性结合。