BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
储存缓冲液：100% Isopropyl Alcohol
UltraBio™ NHS磁珠 (1μm) 是一种预活化的聚合物磁性微球，可以直接用于含氨基的蛋白或多肽的耦联。预活化介质可以根据需要制备成特殊的亲和介质，快速有效地从复杂体系中一步纯化相应的物质。
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使用说明：
一、缓冲液的准备
所用水和缓冲液在使用之前建议用0.22μm或0.45μm滤膜过滤。
清洗液：1mM HCl
偶联液：0.2M NaHCO3，0.5M NaCl，pH8.0
封闭液：0.5M 乙醇胺，0.5M NaCl，pH8.3或0.1M Tris，pH8.5
清洗液1：0.1M 乙酸-乙酸钠，0.5M NaCl，pH3.0
清洗液2：0.1M Tris-HCl，0.5M NaCl，pH8.0
保护液：PBS，0.01%Tween-20，0.02%NaN3
注：偶联液可以选择碳酸盐、磷酸盐等不含氨基的缓冲液体系。缓冲液体系中加入一定浓度的盐离子减少非特异性吸附。
二、样品准备
样品用偶联液溶解，浓度约5-10 mg/mL。
三、抗原偶联
1、取适量的UltraBio™ NHS磁珠 (1μm)，吸去保护液，用1mM HCl清洗液清洗一次，再用偶联液清洗一次。
注：磁珠加入清洗液混匀后立刻放在磁力架上吸附，取走上清。可以选用预冷的溶液快速清洗，减少预活化介质的水解。
2、溶解好的样品加入至清洗好的UltraBio™ NHS磁珠 (1μm) 中。
3、28℃振荡反应2-4h或4℃过夜。
注：确保磁珠悬浮起来，否则会大大影响偶联效率。
4、反应完后收集偶联样品，以便检测偶联效率。去离子水清洗磁珠，加入初始磁珠悬浮液2倍体积的封闭液，28℃振荡反应1h。
注：偶联后无法用紫外吸收测定上清蛋白浓度，建议用电泳或者BCA定量法检测偶联效率。
5、将上述反应体系取出，流干其中的封闭液，用3倍悬浮液体积的去离子水清洗磁珠，清洗液1、去离子水、清洗液2和去离子水重复冲洗2次，然后以10 mg/mL的浓度保存在保护液中，于2-8℃保存。
四、纯化流程
下面以磁珠偶联抗体后，纯化抗原为例，介绍纯化步骤。
1、缓冲液的准备
所用水和缓冲液在使用之前建议用0.22μm或0.45μm滤膜过滤。
平衡/洗杂液：0.15M NaCl，20mM Na2HPO4，pH7.0
洗脱液：0.1M 甘氨酸，pH3.0
中和液：1M Tris-HCl，pH8.5
2、磁珠预处理
将偶联好的磁珠混合均匀，取计算量（根据上样量和磁珠载量计算）的磁珠悬浮液（后文均以100μl为例），转移至离心管中，放置在磁分离器上，静置大约1min，待溶液变澄清后，用移液器吸弃上清液。再将离心管从磁分离器上取下来，加入与所取磁珠悬浮液等体积（100 μl）的平衡液，使用枪头反复吹打5次，将离心管置于磁分离器上，大约1min，待溶液变澄清后，用移液器吸弃上清液，重复洗涤2次。
3、样品吸附
在步骤2预处理的磁珠中加入样品溶液，漩涡振荡均匀，在室温下置于翻转混合仪或者手工轻轻翻转离心管，促使样品和磁珠充分接触 并吸附，混合30min以上（具体时间根据结合效果调整），置于磁分离器上，大约1min，待溶液变澄清后，吸弃上清液。
4、洗杂
向离心管中加入初始磁珠悬浮液5倍体积（500μl）的洗杂液，振荡悬浮，混合1-2min，然后置于磁分离器上，大约1min，待溶液变澄清后，吸弃上清液。该操作重复两次。
5、洗脱
在上述离心管中加入3-5倍悬浮液体积（300-500μl）的洗脱液，用移液器吹打5次，然后在室温下置于翻转混合仪或者手工轻轻翻转离心管，5-10min后，置于磁分离器上，大约1min，待溶液变澄清后，吸取上清液，收集洗脱组分，即为目标蛋白。
6、洗脱组分中和
向洗脱组分中加入洗脱体积十分之一的中和液，调节pH值至7.0-8.0。
7、磁珠保存
使用后的磁珠用1ml洗脱液重悬磁珠，然后置于磁分离器上，大约1min，待溶液变澄清后，吸弃上清液。该操作重复两次。再加入1ml 平衡液，悬浮磁珠，然后置于磁分离器上，大约1min，待溶液变澄清后，吸弃上清液。加入步骤一中的保护液至磁珠浓度为10mg/ml， 置于2～8℃保存。
UltraBio™ NHS Magnetic Beads (1 μm) are pre-activated polymer magnetic microspheres designed for direct coupling with proteins or peptides containing amino groups. This pre-activated medium can be tailored to prepare specialized affinity media, enabling rapid and efficient one-step purification of target substances from complex systems.
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Instructions for Use:
I. Buffer Preparation
Filter all water and buffers through a 0.22 μm or 0.45 μm membrane before use.
Wash Solution: 1 mM HCl
Coupling Buffer: 0.2 M NaHCO₃, 0.5 M NaCl, pH 8.0
Blocking Solution: 0.5 M Ethanolamine, 0.5 M NaCl, pH 8.3 OR 0.1 M Tris, pH 8.5
Wash Buffer 1: 0.1 M Sodium Acetate-Acetic Acid, 0.5 M NaCl, pH 3.0
Wash Buffer 2: 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.0
Storage Solution: PBS, 0.01% Tween-20, 0.02% NaN₃
Note: Coupling buffer can be carbonate, phosphate, or other buffers lacking primary amines. Adding salt ions reduces non-specific adsorption.
II. Sample Preparation
Dissolve the sample in Coupling Buffer to a concentration of approximately 5-10 mg/mL.
III. Antigen Coupling
Take the desired amount of UltraBio™ NHS Magnetic Beads (1 μm). Aspirate the storage solution. Wash once with 1 mM HCl Wash Solution, then once with Coupling Buffer.
*Note: After adding wash solution and mixing, immediately place the tube on the magnetic rack for separation (~1 min), then aspirate the supernatant. Use pre-chilled solutions for rapid washing to minimize hydrolysis of the pre-activated medium.*
Add the dissolved sample to the washed UltraBio™ NHS Magnetic Beads (1 μm).
React with end-over-end rotation at 28°C for 2-4 hours or 4°C overnight.
Note: Ensure the beads remain suspended; settling significantly reduces coupling efficiency.
After reaction, collect the coupling supernatant to assess coupling efficiency. Wash the beads with deionized water. Add twice the initial bead suspension volume of Blocking Solution. React with end-over-end rotation at 28°C for 1 hour.
Note: UV absorbance measurement of supernatant protein concentration is unreliable post-coupling. Use SDS-PAGE or BCA assay to assess coupling efficiency.
Aspirate the blocking solution. Wash the beads sequentially with:
3x suspension volume Deionized Water
1x suspension volume Wash Buffer 1
1x suspension volume Deionized Water
1x suspension volume Wash Buffer 2
1x suspension volume Deionized Water
Repeat this alternating wash cycle once more (2 cycles total). Resuspend the coupled beads in Storage Solution at a concentration of 10 mg/mL. Store at 2-8°C.
IV. Purification Procedure (Example: Antigen Purification using Antibody-Coupled Beads)
Buffer Preparation:
Filter all water and buffers before use.
Equilibration/Wash Buffer: 0.15 M NaCl, 20 mM Na₂HPO₄, pH 7.0
Elution Buffer: 0.1 M Glycine, pH 3.0
Neutralization Buffer: 1 M Tris-HCl, pH 8.5
Bead Pre-equilibration:
Resuspend coupled beads thoroughly. Transfer the calculated volume (e.g., 100 μL) to a microcentrifuge tube.
Place on magnetic rack for ~1 min until solution clears. Aspirate supernatant.
Remove tube from rack. Add an equal volume (e.g., 100 μL) of Equilibration Buffer. Pipette mix 5 times.
Place on magnetic rack for ~1 min. Aspirate supernatant.
Repeat equilibration wash twice (3 washes total).
Sample Binding:
Add sample solution to the pre-equilibrated beads. Vortex-mix briefly.
Incubate with end-over-end rotation (or gentle manual tube inversion) at room temperature for ≥30 min (adjust based on binding efficiency).
Place on magnetic rack for ~1 min. Aspirate and discard supernatant.
Washing:
Add 5x initial bead volume (e.g., 500 μL) of Wash Buffer. Vortex-mix to resuspend.
Incubate with mixing for 1-2 min.
Place on magnetic rack for ~1 min. Aspirate and discard supernatant.
Repeat wash twice (3 washes total).
Elution:
Add 3-5x initial bead volume (e.g., 300-500 μL) of Elution Buffer. Pipette mix 5 times.
Incubate with end-over-end rotation (or gentle manual tube inversion) at room temperature for 5-10 min.
Place on magnetic rack for ~1 min. Transfer the clear supernatant (elution fraction) to a clean tube. This contains the target protein.
Neutralization:
Add 1/10th volume of the elution fraction of Neutralization Buffer to the elution fraction. Mix immediately to adjust pH to 7.0-8.0.
Bead Regeneration & Storage:
Add 1 mL Elution Buffer to the beads. Vortex-mix. Place on magnetic rack for ~1 min. Aspirate supernatant.
Repeat elution wash once (2 washes total).
Add 1 mL Equilibration Buffer. Vortex-mix. Place on magnetic rack for ~1 min. Aspirate supernatant.
Resuspend beads in Storage Solution (Section I) at 10 mg/mL. Store at 2-8°C.
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ25F0826359 |
分析证书 | N1456038 |
| ZJ25F0826358 |
分析证书 | N1456038 |
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