| 货号 (SKU) | 包装规格 | 是否现货 | 价格 | 数量 |
|---|---|---|---|---|
| A751539-500μl |
500μL |
期货  |
| |
| A751539-2ml |
2ml |
期货 |
|
| 产品名称 | UltraBio™ Anti-GST Magnetic Beads (Anti-GST磁珠) |
|---|---|
| 别名 | UltraBio™ 抗 GST 磁珠 |
| 产品介绍 |
阿拉丁生产的UltraBio™ Anti-GST Magnetic Beads，即Anti-GST磁珠，也称Anti-GST免疫磁珠或GST抗体磁珠，是由高品质的GST小鼠单克隆抗体与纳米级氨基磁珠共价偶联而成，可特异性地与动植物或微生物裂解液、血清、腹水等中含有GST标签的蛋白结合，从而用于带有GST标签的融合蛋白或其蛋白复合物的免疫沉淀(Immunoprecipitation, IP)、免疫共沉淀(Co-IP)或纯化。GST标签、Flag标签、Myc标签(Myc-tag)、HA标签(HA-tag) 和His标签(His-tag)等是表达载体上最常见的一些标签，通过与这些标签的融合表达可以非常方便地检测目的蛋白及与目的蛋白相互结合的蛋白，也可以非常方便地用于目的蛋白的纯化。GST标签，即谷胱甘肽S-转移酶(Glutathione S-transferase)标签，GST本身是一个在解毒过程中起到重要作用的转移酶，分子量约为26kDa，可融合在蛋白的N端或者C端，在大肠杆菌中常融合于N端。GST标签具有以下优点：能增加外源蛋白的可溶性和表达量；可在不同的宿主如大肠杆菌和酵母中表达，适用范围广；可很好保留了蛋白的抗原性和生物活性，有助于保护重组蛋白免受胞外蛋白酶的降解并提高其稳定性；如果在GST标签和目的蛋白之间含有位点特异性的蛋白酶识别位点，如PreScission Protease、TEV Protease或Thrombin等，则可用相应的蛋白酶切除GST标签，方便去除；高特异性，纯化方便且温和；GST标签作为标签蛋白，后续通过GST抗体或Anti-GST磁珠、UltraBio™ GST-tag Purification Resin、GST标签蛋白纯化试剂盒等即可对目的基因的表达、定位及功能进行检测或对目的蛋白进行纯化、免疫沉淀或免疫共沉淀等。基于以上优点，GST标签已被广泛应用于蛋白表达、纯化、鉴定、相互作用和功能等多方面的研究。一般使用GST纯化柱对GST标签蛋白进行纯化，但对于带有GST标签的融合蛋白或蛋白复合物的少量纯化或免疫沉淀等应用，Anti-GST磁珠更简单、便捷。UltraBio™ Anti-GST Magnetic Beads (Anti-GST磁珠)可特异性地结合GST标签融合蛋白，并可以借助磁力架等磁分离设备非常便捷地应用于带有GST标签的融合蛋白或其蛋白复合物的免疫沉淀或纯化等实验。本产品进行免疫沉淀的流程参考图1。图1. 阿拉丁的UltraBio™ Anti-GST Magnetic Beads (Anti-GST磁珠)免疫沉淀流程图。本产品特异性强、靶蛋白结合量高。与国内外大多数的同类产品相比，本产品抗体结合密度高，对带有GST标签蛋白的结合具有很强的特异性，并且本产品磁珠粒径小，不易产生非特异吸附。本产品每毫升磁珠悬浊液含约10mg磁珠，含有不少于0.6mg GST抗体，通常可结合不少于0.6mg GST标签融合蛋白，具体的最大结合量和标签蛋白的分子量大小等相关。每500微升样品，通常仅需使用10-20微升磁珠悬浊液，就可以高效地进行免疫沉淀实验。本产品可结合多种形式的GST标签蛋白。本产品可特异性地结合甲硫氨酸修饰的N端GST融合蛋白(Met-GST-Protein)、N端GST融合蛋白、C端GST融合蛋白(Protein-GST)。本产品结合目的蛋白速度快。本产品使用了纳米级磁珠(~200nm)，具有超大的比表面积，便于抗体和抗原的快速有效结合。通常10分钟内即可完成抗原吸附的过程，30分钟内完成目的蛋白免疫沉淀操作。缩短操作时间可以有效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。本产品可选择多种洗脱方法。本产品可以根据目的蛋白的结构、生物学功能及后续应用的要求等，使用多种洗脱方法，包括SDS-PAGE上样缓冲液和酸性等洗脱液进行洗脱。本产品用于GST融合蛋白的免疫沉淀效果参考图2。图2. UltraBio™ Anti-GST Magnetic Beads (Anti-GST磁珠)用于GST融合蛋白的免疫沉淀效果图。样品1为Input，即原核表达的GST-tag融合蛋白全细胞裂解液；样品2为Mouse IgG Magnetic Beads (小鼠IgG免疫磁珠) 免疫沉淀后经SDS-PAGE蛋白上样缓冲液(1X) 洗脱后的样品，该Mouse IgG是正常的小鼠IgG (Normal Mouse IgG)，为阴性对照；样品3为本磁珠免疫沉淀后的样品，使用SDS-PAGE蛋白上样缓冲液(1X)洗脱。使用SDS-PAGE蛋白上样缓冲液(1X)洗脱后可以检测到GST抗体的轻重链。Western印迹成像由Imager 600化学发光成像系统完成。实际结果会因实验条件、检测仪器等的不同而存在差异，图中数据仅供参考。本产品的主要指标如下表： 注意事项： 由于GST标签本身的分子量较大，约26kDa，与目的基因形成融合蛋白后分子量更大，而且融合蛋白的结构复杂性可能会影响Anti-GST磁珠对GST蛋白的识别，建议通过预实验进行免疫沉淀的验证或对裂解条件进行一定的调整。由于GST-tag与GST抗体的结合力非常强，酸性洗脱的效果可能比较差，建议优先使用SDS-PAGE上样缓冲液洗脱法。本产品需维持pH为6-8，避免高速离心和干燥；请勿长时间将磁珠置于磁场中，否则可能会引起磁珠聚团。本产品使用前要适当充分重悬，即颠倒若干次使磁珠混合均匀，混匀操作须轻柔，不宜剧烈涡旋震荡等，避免抗体变性等。在免疫沉淀或纯化时，建议设置阳性和阴性对照组。蛋白样品收集后宜尽快完成纯化工作，并应始终放置在4℃或冰浴，以减缓蛋白降解或变性。为有效抑制蛋白降解，可以在蛋白样品中添加适量的蛋白酶抑制剂混合物，例如阿拉丁的P1005/P1006蛋白酶抑制剂混合物(通用型)、P1048/P1049 蛋白酶磷酸酶抑制剂混合物(通用型, 质谱兼容, 50X)、P1010/P1011 蛋白酶抑制剂混合物(哺乳动物样品抽提用, 100X)、P1050/P1051 蛋白酶磷酸酶抑制剂混合物(哺乳动物样品抽提用, 50X)等。如果使用真空泵等仪器吸取上清液，须注意真空泵的吸液强度，以免吸力过大而吸取到聚集的磁珠。酸性溶液洗脱时磁珠可能会发生聚集，属于正常现象，不影响磁珠的正常使用。0.1%的非离子型去垢剂(如Triton X-100、Tween-20或NP-40)可有效防止磁珠聚集，并且不会影响磁珠的抗体结合效率。高浓度的DTT、巯基乙醇、盐酸胍等对本产品与标签蛋白的结合可能有一定影响，但Western及IP细胞裂解液、RIPA裂解液或NP-40裂解液等都完全适用。阿拉丁生产的不同裂解液的主要特点和差异，以及如何选择裂解液可参考我们的相关网页：http://www.aladdin-e.com/support/lysis-buffer.htm。本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。为了您的安全和健康，请穿实验服并戴一次性手套操作。使用说明： 1.样品的制备。a.选择合适的裂解液，用于制备细胞或组织的裂解液。优先推荐选择阿拉丁生产的P0013 Western及IP细胞裂解液用于细胞或组织样品的裂解。根据特定的实验目的，如有必要，也可以使用阿拉丁生产的P0013B RIPA裂解液(强)、P0013C RIPA裂解液(中)或P0013D RIPA裂解液(弱)用于样品的制备。如果使用自行配制的或其它公司生产的裂解液，需要确保裂解液的pH为6-8。注：由于GST标签本身的分子量较大，约26kDa，与目的基因形成融合蛋白后分子量更大，而且融合蛋白的结构复杂性可能会影响Anti-GST磁珠对GST蛋白的识别，建议通过预实验进行免疫沉淀的验证或对裂解条件进行一定的调整。b.具体的细胞或组织样品裂解的制备步骤请参考裂解液的使用说明。制备好的裂解液上清宜置于冰上或4℃存放，随后即可用于免疫沉淀或免疫共沉淀、标签蛋白的纯化等操作。新鲜制备好的样品，建议尽量当天完成免疫沉淀等后续操作，但如果样品不能当天使用，也可以适当分装后-80℃冻存。2.Anti-GST磁珠的准备。由于Anti-GST磁珠储存在特殊保护液中，所以需要在加入样品前适当洗涤。a.用移液器轻轻吹打重悬Anti-GST磁珠，按照每500μl样品10μl或20μl磁珠悬浊液，取适量Anti-GST磁珠至一洁净离心管中，加入1X TBS 至最终体积为约0.5ml。b.用移液器轻轻吹打并充分重悬Anti-GST磁珠。置于磁力架(FMS012/FMS024)上分离10秒，去除上清。重复上述步骤两次。c.按照初始体积的量，用1X TBS 重悬Anti-GST磁珠。3.免疫沉淀(Immunoprecipitation, IP):a.加入磁珠与孵育。按照每500μl蛋白样品加入10μl或20μl磁珠悬浊液的比例加入Anti-GST磁珠，置于侧摆摇床或旋转混合仪上，室温孵育2小时或4℃孵育过夜。注：孵育过程中，如果磁珠发生聚团或呈片状属正常现象，不会影响实验结果。b.磁分离。孵育完毕后，置于磁力架上分离10秒，去除上清。注：可保留部分上清液，用于检测免疫沉淀的效果。c.洗涤。加入500μl的1X TBS，用移液器轻轻吹打重悬Anti-GST磁珠。置于磁力架上分离10秒，去除上清。重复洗涤三次。注：也可以通过检测洗涤得到的洗涤液的OD280来判断是否洗涤完全，若OD280大于0.05，应适当增加洗涤次数。4.洗脱：根据标签蛋白的特点及后续实验要求，可以选择如下2种方法之一进行洗脱。a.SDS-PAGE上样缓冲液洗脱法：本方法为变性法，得到的蛋白样品适合SDS-PAGE电泳或WB检测。(a)SDS-PAGE上样缓冲液的配制：可以直接使用阿拉丁生产的P0015A SDS-PAGE蛋白上样缓冲液(1X)，或使用阿拉丁生产的P0015 SDS-PAGE蛋白上样缓冲液(5X)或自行参考《分子克隆》等配制5X或2X的SDS-PAGE蛋白上样缓冲液，然后加入水配制成1X的SDS-PAGE蛋白上样缓冲液。通常SDS-PAGE蛋白上样缓冲液含有DTT等还原剂，其洗脱得到的蛋白样品中会含有GST抗体的轻链和重链。(b)每10-20μl原始磁珠体积的磁珠，加入100μl 1X SDS-PAGE上样缓冲液，95℃加热5分钟。(c)置于磁力架上分离10秒，取上清进行SDS-PAGE电泳或Western检测。b.酸性洗脱法：本方法为非变性法，比较快速且高效。洗脱后的蛋白保持原有的生物活性，便于后续分析检测。(a)溶液的配制：酸性洗脱液(0.1M Glycine-HCl, pH3.0)，中和液(0.5M Tris-HCl, pH7.4, 1.5M NaCl)。(b)每10-20μl原始磁珠体积，加入100μl酸性洗脱液，混匀后置于侧摆摇床或旋转混合仪上，室温孵育5分钟。注：孵育时间不宜超过15分钟。注：洗脱液的体积可以酌情适当调整，同时须注意后续的中和液体积也需要作相应调整。(c)孵育完毕后，置于磁力架上分离10秒，将上清转移到新的离心管中，并加入10μl中和液，适当混匀。(d)为了获得最大的洗脱效率，可重复步骤b和c，并将相同样品合并。(e)洗脱并中和的GST标签蛋白置于4℃待用，或者-20℃或-80℃长期保存。注1：酸性洗脱法虽然高效，但仍可能低于SDS-PAGE上样缓冲液洗脱法。注2：由于目的蛋白的差异可能对酸性洗脱法的洗脱效率有一定的影响，如果对洗脱效率的要求比较高，可对酸性洗脱液的pH在2.5-3.1之间进行一定的调整，相应的中和液的pH值或量也要进行一定的调整，例如100μl酸性洗脱液(0.1M Glycine-HCl, pH2.8)和15μl中和液(1M Tris-HCl, pH8.5)。常见问题： Problem Possible Causes Solution Very few or no GST‐tagged protein exists in the eluate. Protein is not completely eluted. Change elution methods. No target protein expressed. Make sure the protein of interest contains the GST-tag by Western blot or dot blot analyses. Very low protein expression level. 1. Use larger volume of cell lysate. 2. Optimize expression conditions to raise the protein expression level. Washes are too stringent. Reduce the time and number of washes. Incubation times are inadequate. Increase the incubation time. Interfering substance is present in sample. Lysates containing high concentration of DTT, 2-mercaptoethanol, or other reducing agents may destroy antibody function, and must be avoided. Detection system is inadequate. If Western blot detection is used: 1. Check primary and secondary antibodies using proper controls to confirm binding and reactivity.2. Verify that the transfer was adequate by using prestained protein marker or staining the membrane with Ponceau S.3. Use fresh detection substrate or try a different detection system. Background is too high. Proteins bind nonspecifically to the Anti-GST monoclonal antibody, insufficient washing on magnetic beads, or the microcentrifuge tubes. 1. Pre-clear lysate with Mouse IgG Magnetic Beads to remove nonspecific binding proteins. 2. After suspending beads for the final wash, transfer entire sample to a clean microcentrifuge tube before centrifugation. Washes are insufficient. 1.Increase the number of washes.2.Prolong duration of the washes, incubating each wash for at least 15 minutes.3.Increase the salt and/or detergent concentrations in the wash solutions.4.Centrifuge at lower speed to avoid nonspecific trapping of denatured proteins. Multiple protein bands found in the eluate. The protein is not stable at room temperature. Purify the target protein at lower temperature, such as 4℃. Protein degradation due to proteases activity during purification process. Add protease inhibitors to cell lysate. Non‐specific binding. 1.Prepare cell lysate again.2.Add additional wash steps.Aladdin's UltraBio™ Anti-GST Magnetic Beads are nano-scale amino magnetic beads covalently coupled with high-quality mouse monoclonal antibody that recognizes the GST tag specifically. This product can specifically bind GST-tagged proteins expressed in animals, plants, and microorganisms, and is recommended to use for immunoprecipitation (IP), co-immunoprecipitation (Co-IP), and protein purification.GST (glutathione S-transferase) plays an important role in the detoxification process, with a molecular weight of approximately 26kDa. It can be fused to the N-terminus or C-terminus of a target protein for convenient purification and detection. There are several advantages for using the GST as a tag. It can act as a chaperone to facilitate protein folding and increases the solubility of GST fusion proteins. It can be expressed in different hosts such as E. coli and yeast, and has a wide range of applications. It can well retain the antigenicity and biological activity of target proteins, which helps protect the recombinant protein from degradation by extracellular proteases. The GST tag can be easily removed if there is a site-specific protease recognition site between the GST tag and the target protein, such as PreScission Protease, TEV Protease, or Thrombin. The GST fusion protein can be purified specifically and conveniently by using Aladdin's GST Antibody (AH158), Anti-GST Magnetic Beads, UltraBio™ GST-tag Purification Resin, or GST Tag Protein Purification Kit . The fusion protein can also be easily detected, characterized, and used for IP or Co-IP analysis. Therefore, GST tags have been widely used in protein expression, purification, identification, protein-protein interaction, and functional studies. UltraBio™ Anti-GST Magnetic Beads specifically bind with GST fusion proteins. With a magnetic stand, this product can be conveniently used for protein purification and immunoassays. Please refer to Figure 1 for the workflow of IP assays with this product.Figure 1. Workflow of Immunoprecipitation (IP) with Aladdin's UltraBio™ Anti-GST Magnetic Beads .High specificity and binding capacity Precautions： As the molecular weight of GST tag is relatively large (~26kDa), the molecular weight of the fusion protein is large and the structural complexity of the fusion protein may affect the recognition of the GST fusion protein by the Anti-GST magnetic beads. We recommend validating or adjusting conditions by preliminary experiments.As GST-tag binds very strongly to GST antibody, acidic elution may be less effective. It is recommended to use SDS-PAGE loading buffer for elution.The pH of Mag™ Anti-GST Magnetic Beads should be maintained at 6-8. Do not centrifuge, dry, or freeze the magnetic beads. Long-time exposure of the beads to a magnetic field will causes beads to aggregate.Resuspend Mag™ Anti-GST Magnetic Beads evenly in solution by gentle pipetting prior to use. Do not vortex.We recommend including both negative and positive controls when performing IP assays.Protein samples should be purified after collection as soon as possible and kept at 4℃ or on ice to minimize protein degradation or denaturation. Protein degradation can also be inhibited by adding appropriate protease and phosphatase inhibitors (, P1005/P1006, P1048/P1049, P1010/P1011, P1050/P1051).If using a vacuum pump to aspirate supernatant, the strength of the vacuum pump should be controlled properly to avoid the aspiration of magnetic beads.It is normal to see magnetic beads aggregate when an acidic solution is used for the elution, which can be prevented effectively by containing 0.1% non-ionic detergent (such as Triton X-100, Tween-20, or NP-40) without affecting the performance of this product.High concentrations of DTT, mercaptoethanol, or anidin hydrochloride might affect the binding of the target protein to this product, but it is compatible with ’s Cell Lysis Buffer for Western and IP , RIPA Lysis Buffer or NP-40 Lysis Buffer . For detailed information about the Lysis Buffers produced by , please refer to the following webpage:http://www.aladdin-e.com/support/lysis-buffer.htm.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use： 1.Preparation of protein samplesa.Lysis cells or tissues with an appropriate lysis buffer, such as Protein A Magnetic Beads1mlP2102-5ml Protein A Magnetic Beads5mlP2105-1ml Protein G Magnetic Beads1mlP2105-5ml Protein G Magnetic Beads5mlP2108-1ml Protein A+G Magnetic Beads1mlP2108-5ml Protein A+G Magnetic Beads5mlP2115-0.5ml Anti-Flag Magnetic Beads0.5mlP2115-2ml Anti-Flag Magnetic Beads2mlP2118-0.5ml Anti-Myc Magnetic Beads0.5mlP2118-2ml Anti-Myc Magnetic Beads2mlP2121-0.5ml Anti-HA Magnetic Beads0.5mlP2121-2ml Anti-HA Magnetic Beads2mlP2132-0.5ml Anti-GFP Magnetic Beads 0.5mlP2132-2ml Anti-GFP Magnetic Beads 2mlP2135-0.5ml Anti-His Magnetic Beads 0.5mlP2135-2ml Anti-His Magnetic Beads 2mlP2138-0.5ml Anti-GST Magnetic Beads0.5mlP2138-2ml Anti-GST Magnetic Beads2mlP2141-0.5ml Anti-V5 Magnetic Beads0.5mlP2141-2ml Anti-V5 Magnetic Beads2mlP2151-200μl Streptavidin Magnetic Beads200μlP2151-1ml Streptavidin Magnetic Beads1mlP2151-5ml Streptavidin Magnetic Beads5mlP2171-1ml Mouse IgG Magnetic Beads1mlP2171-5ml Mouse IgG Magnetic Beads5mlP2173-1ml Rabbit IgG Magnetic Beads 1mlP2173-5ml Rabbit IgG Magnetic Beads 5ml |
| 储存温度 | -20°C储存 |
|---|---|
| 运输条件 | 超低温冰袋运输 |
| 稳定性与储存 | -20ºC保存，两年有效。4ºC保存，至少三个月内有效。 |
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