计算溶液所需的质量、体积或浓度。
MBP标签蛋白纯化试剂盒 (pH 7.4)
有货
为什么选择此级别
BioReagent,用于蛋白分析 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
2-8°C储存,室温,禁止冷冻。低温运输,禁止冷冻 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
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。
概述
Dextrin亲和层析树脂是一种将糊精键合在琼脂糖基球上形成的亲和层析分离介质。糊精配基可与麦芽糖结合蛋白(MBP)特异性结合，因而可纯化带MBP标签的融合蛋白。本产品可在接近生理条件下纯化目标蛋白，可最大程度保留目标蛋白的活性。MBP标签较大，分子量约为42 kDa。MBP标签的主要作用有促进目标蛋白正确折叠，增加蛋白的表达水平，提高目标蛋白的可溶性。同时带MBP标签的融合蛋白特定的亲和填料，MBP标签在大肠杆菌体系中经常使用，亲和层析后通常利用特异性酶去除标签。该树脂实际凝胶每毫升可承载3-5 mg MBP融合蛋白。Dextrin Resin凝胶的颗粒直径为45-165 μm，可耐受的最大压力为0.3 MPa，Dextrin亲和层析树脂储存在20%乙醇中，凝胶和保护液的体积比为1:1，10 mL总体积中5 mL为凝胶、5mL为液体。使用时宜把凝胶充分重悬后再吸取。
产品特点
1. 快速、高效：本试剂盒使用了MBP标签蛋白纯化树脂，挂柱效率高，缩短操作时间的同时可以有效的避免在长时间操作过程中目的蛋白的降解或变性，充分保证目的蛋白的活性。
2. 便捷：提供了MBP标签蛋白纯化时所需的相关试剂和亲和层析柱空柱管 ，为MBP标签蛋白的纯化带来了极大的便利。
3. 特异性强： 糊精配基可与麦芽糖结合蛋白(MBP)特异性结合,可进行一步纯化。
4. 不影响蛋白活性：采用麦芽糖温和洗脱，无去污剂和变性剂对蛋白活性的影响。
使用方法
一、 样品准备
1、 细菌或酵母表达的可溶性蛋白
(1) 挑取单菌落到含有适合抗性的培养基中，根据载体使用说明，加入相应浓度的诱导剂诱导相应的时间。
(2) 表达结束后，将培养液转移到离心瓶中，7000 rpm (7500 ×g)，离心15 min收集菌体，然后按照菌体与裂解液比例为1 : 10 (W/V) 加入裂解液，根据蛋白易降解性质可选择性加入终浓度为1 mM的PMSF，也可加入其他蛋白酶抑制剂，但不能影响目的蛋白与填料的结合。根据需求可加入溶菌酶(如果表达的宿主细胞内含pLysS或pLysE，可以不加溶菌酶)。
(3) 将菌体沉淀悬浮起来(如果菌液浓度高，也可考虑加入10 μg/mL RNaseA和5 μg/mL DNaseI)，混匀，放置于冰上，然后冰水混合物上超声破碎细胞，至菌液基本保持澄清。
(4) 将澄清的破碎液转移至离心管中，10000 rpm (15000 ×g)，4℃离心20-30 min。取上清，置于冰上备用，进行下一步纯化。
2、 酵母、昆虫和哺乳细胞分泌表达可溶性蛋白
将细胞培养液转移至离心瓶，5000 rpm (3800 ×g)，离心10 min，收集上清，可直接上柱使用。对于大量体积的上清，需加入硫酸铵沉淀浓缩后，蛋白还需用平衡液透析后才能加入柱子。
二、 缓冲液的准备
含麦芽糖洗脱缓冲液的配制：洗脱缓冲液不含麦芽糖，添加360 mg麦芽糖至100 mL洗脱缓冲液中，以配制含10 mM麦芽糖洗脱缓冲液，配制完后可用0.22 μm或0.45 μm滤器过滤，并将溶液保存在2-8℃。可根据实验使用量配制，宜现配现用。
三、 样品纯化
样品在上样前建议离心或用0.22 μm或0.45 μm滤膜过滤，减少杂质，提高蛋白纯化效率和防止堵塞柱子。缓冲液在使用前最好用0.22 μm或者0.45 μm滤膜过滤。
A. 重力柱法纯化
1、 重力柱的装填
(1) 取层析空柱，装入下垫片，加入适量纯水润洗柱管和垫片，关闭下出口。
(2) 将Dextrin亲和树脂充分混匀，用移液器吸取适量浆液加入重力柱中(填料实际体积占悬液的一半，如吸取1 mL悬液，其实际体积为0.5 mL，则最大载量约为2.5 mg)，打开下出口流干保护液。
(3) 加入适量纯水冲洗填料，待柱管中液体重力流干后，关闭下出口。
(4) 加入润洗后的上垫片，确保垫片与填料之间没有空隙，且保持水平。
(5) 装填好的重力柱可以直接加入Lysis Buffer/Wash Buffer进行平衡，暂不使用时则加入保护液，4℃保存。
2、 平衡
将装填好Dextrin亲和层析树脂的重力柱用2-5 CV(CV是指沉降后的树脂体积，以下内容提到的CV不再做此注解)裂解缓冲液或洗杂缓冲液进行平衡，重复2-3次。
3、 上样
将样品加到平衡好的重力柱中，样品缓慢流出，保证样品和填料充分接触，收集流出液，可以反复上样增加结合效率 (如果目标蛋白不稳定，建议样品置于冰上)。
4、 洗杂
使用5-10 CV的洗杂缓冲液洗涤柱子，去除非特异性吸附的杂蛋白，收集洗杂液。洗柱及下一步洗脱过程中可以用Bradford法简单快速地检测每次洗涤液和洗脱液中的蛋白含量，从而考虑增加或减少洗涤和洗脱的次数。
5、 洗脱
使用5-10 CV的含麦芽糖洗脱缓冲液洗脱目的蛋白。分段收集，每1-5个CV收集一管，分别检测，既可以保证所有结合的目的蛋白被洗脱，又可以得到高纯度和高浓度的蛋白。
B. 孵育法纯化
1、 树脂准备
根据纯化的样品量，取适量Dextrin亲和层析树脂加入离心管中，1000 rpm离心1 min，小心的吸弃上清(保持低速离心，以免损坏树脂)；也可加入重力柱中，流干保护液。
2、 平衡
向离心管中加入5 CV裂解缓冲液或洗杂缓冲液，轻柔上下翻动混匀，1000 rpm 离心1 min后，小心的去除上清；如使用重力柱，则直接在重力柱中清洗，直接重力流干洗涤液；重复两次以上。
3、 树脂结合目的蛋白
将准备好的样品加入到平衡好的树脂中，封闭离心管或重力柱管，旋转振荡孵育，室温30 min-2 h或2-8℃ 2-4 h，可适当延长时间，使混合液与树脂充分混匀(如果目标蛋白不稳定，建议置于2-8℃环境中结合)。1000 rpm 离心1 min后，小心的吸出上清，将上清留存进行检测分析。
4、 洗杂
使用 5 CV的洗杂缓冲液洗涤柱子，轻柔上下翻动混匀，1000 rpm 离心1 min后，小心的吸出上清，将上清留存进行检测分析。重复3-5次。如使用重力柱，则直接在重力柱中清洗，直接重力流干洗涤液。
5、 洗脱
加入3-5 CV的含麦芽糖洗脱缓冲液进行洗脱，室温孵育5min，1000 rpm离心1 min或重力柱管收集洗脱液，可重复2-3次。
上述步骤洗脱结束后，用洗杂液冲洗3 CV，然后用纯水冲洗5 CV，再用保护液冲洗2 CV，然后将树脂置于2-8℃保存。
四、 样品检测与填料清洗
1、 SDS-PAGE 检测
将纯化过程中得到的样品 (包括原始样品、流出组分、洗杂及洗脱组分等) 利用 SDS-PAGE 进行检测，判定其纯化效果。
2、 填料清洗
洗脱目的蛋白后的柱子用纯水清洗3-5个柱体积，并用0.5 M NaOH再生3-5个柱体积，再用纯水清洗至中性，用20%乙醇保存柱子，或用结合缓冲液平衡后进行下一次纯化，建议流速为75~150 cm/h。
注意事项
1. 请勿冷冻保存Dextrin亲和层析树脂；
2. Dextrin亲和层析树脂储存在含20%乙醇中，凝胶和保护液的体积比为1:1；
3. Dextrin亲和层析树脂使用前一定要充分颠倒若干次，使琼脂糖珠混合均匀；
4. 本产品仅作科研用途；
5. 为了您的安全和健康，请穿实验服并佩戴一次性手套操作。
Dextrin affinity chromatography resin is an affinity chromatography separation medium formed by covalently coupling dextrin ligands to agarose beads. The dextrin ligand specifically binds to maltose-binding protein (MBP), enabling the purification of MBP-tagged fusion proteins. This product allows target protein purification under near-physiological conditions, maximally preserving the biological activity of the target protein. The MBP tag is relatively large, with a molecular weight of approximately 42 kDa. Its main functions include promoting the correct folding of the target protein, enhancing protein expression levels, and improving the solubility of the target protein. Meanwhile, MBP-tagged fusion proteins are compatible with specific affinity resins, and the MBP tag is widely used in Escherichia coli expression systems. After affinity chromatography, the tag is usually removed by site-specific proteases. The binding capacity of this resin is 3-5 mg of MBP fusion protein per milliliter of settled gel. The particle size of Dextrin Resin ranges from 45 to 165 μm, and the maximum withstand pressure is 0.3 MPa. Dextrin affinity chromatography resin is supplied in 20% ethanol, with a 1:1 volume ratio of gel to storage solution. For a total volume of 10 mL, 5 mL is settled gel and 5 mL is storage solution. The gel should be fully resuspended before pipetting during use.
Product Features
1. Fast and Efficient: This kit utilizes MBP-tagged protein purification resin with high binding efficiency. It shortens the operation time and effectively prevents degradation or denaturation of the target protein during prolonged handling, thereby fully preserving the activity of the target protein.
2. Convenient: All necessary reagents and empty affinity chromatography columns for MBP-tagged protein purification are provided, greatly facilitating the purification process of MBP-tagged proteins.
3. High Specificity: The dextrin ligand specifically binds to maltose-binding protein (MBP), enabling one-step purification of the target protein.
4. No Impact on Protein Activity: Mild elution is performed using maltose, without the use of detergents or denaturants that would affect protein activity.
Instruction for use
1 Sample Preparation
1.1 Soluble Protein Expressed in Bacteria or Yeast
1.1.1 Pick a single colony into medium containing the appropriate antibiotic. Induce expression with inducer at the recommended concentration and duration according to the vector instructions.
1.1.2 After expression, transfer the culture to a centrifuge bottle and centrifuge at 7000 rpm (7500 ×g) for 15 min to harvest cells. Resuspend the cell pellet in Native Lysis Buffer at a ratio of 1:10 (w/v). Optionally add PMSF to a final concentration of 1 mM or other protease inhibitors if the protein is prone to degradation, provided they do not interfere with binding to the resin. Lysozyme may be added as needed (not required if the host strain contains pLysS or pLysE).
1.1.3 Fully resuspend the cell pellet. If the cell suspension is concentrated, add 10 μg/mL RNase A and 5 μg/mL DNase I. Mix well, keep on ice, and lyse by sonication in an ice-water bath until the lysate becomes clear.
1.1.4 Transfer the lysate to a centrifuge tube and centrifuge at 10000 rpm (15000 ×g) at 4 °C for 20-30 min. Collect the supernatant, keep on ice, and proceed to purification.
1.2 Soluble Protein Expressed in Yeast, Insect, or Mammalian Cells
Transfer the cell culture to a centrifuge bottle and centrifuge at 5000 rpm (3800 ×g) for 10 min. Collect the supernatant, which may be loaded directly. For large volumes, concentrate by ammonium sulfate precipitation and dialyze against equilibration buffer before loading.
2 Buffer Preparation
Preparation of Elution Buffer Containing Maltose Monohydrate: The elution buffer is supplied without maltose. Add 360 mg of maltose monohydrate to 100 mL of elution buffer to prepare elution buffer containing 10 mM maltose. After preparation, filter the solution through a 0.22 μm or 0.45 μm filter and store at 2-8°C. Prepare according to experimental consumption; fresh preparation is recommended.
3 Sample Purification
Before loading, samples are recommended to be centrifuged or filtered through a 0.22 μm or 0.45 μm filter to reduce impurities, improve purification efficiency, and prevent column clogging. Buffers are also recommended to be filtered through a 0.22 μm or 0.45 μm filter before use.
3.1 Gravity Column Purification
3.1.1 Column Packing
3.1.1.1 Take an empty gravity column, insert the bottom frit, rinse with purified water, and close the bottom outlet.
3.1.1.2 Mix the Dextrin Resin thoroughly and pipette the slurry into the gravity column. The settled resin volume is approximately half the slurry volume (e.g., 1 mL slurry yields ~0.5 mL resin, with a maximum capacity of ~10 mg). Open the outlet to drain the storage solution.
3.1.1.3 Wash the resin with purified water. Drain completely and close the outlet.
3.1.1.4 Insert the top frit, ensuring no air gap between frit and resin and that the frit is level.
3.1.1.5 The packed column may be equilibrated immediately with lysis buffer/wash buffer. If not used immediately, add storage solution and store at 4 °C.
3.1.2 Equilibration
Equilibrate the column with 2-5 CV Lysis Buffer/Wash Buffer (CV = settled resin volume; this definition applies throughout the protocol). Repeat 2-3 times.
3.1.3 Sample Loading
Apply the sample to the equilibrated column. Allow the sample to flow slowly to ensure sufficient binding. Collect the flow-through; reloading may increase binding efficiency. Keep samples on ice if the target protein is unstable.
3.1.4 Washing
Wash with 5-10 CV Wash Buffer to remove non‑specifically bound proteins. Collect the wash fraction.During washing and subsequent elution, protein concentration in each wash and elution fraction can be rapidly monitored using the Bradford method to adjust the number of washing and elution steps as needed.
3.1.5 Elution
Elute the target protein with 5-10 CV Elution Buffer Containing Maltose Monohydrate. Collect fractions every 1-5 CV and analyze individually to ensure complete elution and obtain highly pure, concentrated protein. After elution, wash with 3 CV equilibration buffer, 5 CV purified water, and 2 CV storage solution. Store the resin at 2-8 °C.
3.2 Batch Purification
3.2.1 Resin Preparation
Transfer an appropriate volume of Dextrin Resin into a centrifuge tube. Centrifuge at 1000 rpm for 1 min and carefully remove the supernatant (use low speed to avoid damaging the resin). Alternatively, drain the storage solution in a gravity column.
3.2.2 Resin Equilibration
Add 5 CV Lysis Buffer/Wash Buffer, mix gently by inversion, centrifuge at 1000 rpm for 1 min, and carefully remove the supernatant. Repeat at least twice. For gravity columns, wash and drain by gravity.
3.2.3 Binding
Add the prepared sample to the equilibrated resin. Seal the tube or column and mix by rotation. Incubate at room temperature for 30 min-2 h or at 2-8 °C for 2-4 h (longer if needed). Keep at 2-8 °C if the protein is unstable. Centrifuge at 1000 rpm for 1 min and retain the supernatant for analysis.
3.2.4 Washing
Wash with 5 CV Wash Buffer, mix gently, centrifuge at 1000 rpm for 1 min, and retain the supernatant. Repeat 3-5 times. For gravity columns, wash and drain by gravity.
3.2.5 Elution
Elute with 3-5 CV Elution Buffer Containing Maltose Monohydrate. Incubate at room temperature for 5 min. Centrifuge at 1000 rpm for 1 min or collect by gravity flow. Repeat 2-3 times. After elution, wash with 3 CV equilibration buffer, 5 CV purified water, and 2 CV storage solution. Store the resin at 2-8 °C.
4 Sample Detection and Resin Cleaning
4.1 SDS-PAGE
Analyze samples from the purification process (starting material, flow-through, wash, and elution fractions) by SDS-PAGE to evaluate purification efficiency.
4.2 Resin Cleaning
After eluting the target protein, the column is washed with pure water for 3-5 column volumes, then regenerated with 0.5 M NaOH for 3-5 column volumes. It is then washed with pure water until neutral, and stored in 20% ethanol or equilibrated with the binding buffer for the next purification. The recommended flow rate is 75-150 cm/h.
Matters needing attention
1. Do not freeze the Dextrin Resin.
2. Dextrin Resin is supplied in 1×PBS containing 20% ethanol, with a 1:1 ratio of settled resin to storage solution.
3. The resin must be thoroughly resuspended by repeated inversion before use to ensure uniform agarose bead suspension.
4. This product is for research use only.
5. For your safety and health, wear a lab coat and disposable gloves during all experiments.
规格
别名
MBP标签蛋白纯化试剂盒 | MBP标签纯化试剂盒
英文别名
MBP-Tag Protein Purification Kit
规格或纯度
BioReagent, 用于蛋白分析
英文名称
MBP-Tag Protein Purification Kit (pH 7.4)
应用
Protein purification
储存与运输
储存条件
2-8°C储存,室温,禁止冷冻
运输条件
低温运输,禁止冷冻
稳定性与储存
各组分在相应的储存条件下保质期1年。
组分与储存
| M15058027 | Components | 10T | Storage | Quantity Per Test |
| M15058027A | Dextrin亲和层析树脂 | 10 mL | 2-8°C, Do not freeze | 1 mL |
| M15058027B | 裂解缓冲液 | 180 mL | 2-8°C | 18 mL |
| M15058027C | 洗杂缓冲液 | 100 mL | 2-8°C | 10 mL |
| M15058027D | 洗脱缓冲液 | 100 mL | 2-8°C | 10 mL |
| M15058027E | 麦芽糖 | 360 mg | Room Temperature | 36 mg |
| M15058027F | 层析空柱，3 mL(盖，塞，膜) | 10 EA | Room Temperature | 1 EA |
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
输入批号以搜索分析图谱:
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0737016 |
分析证书 | M15058027 |
技术文档和文章
溶液计算器
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