UltraBio™ Rabbit IgG Magnetic Beads (兔IgG磁珠)

R751580-1ml

1ml

阿拉丁生产的UltraBio™ Rabbit IgG Magnetic Beads，即兔IgG磁珠，也称兔IgG免疫磁珠、Rabbit Normal IgG Magnetic beads、兔正常IgG磁珠或兔正常IgG免疫磁珠，是由高品质的正常兔IgG与纳米级氨基磁珠共价偶联而成，通常用作免疫沉淀(Immunoprecipitation, IP)、免疫共沉淀(Co-IP)、染色质免疫沉淀(Chromatin immunoprecipitation, ChIP)等抗体相关实验时兔来源抗体磁珠的对照IgG磁珠。本兔IgG磁珠中的兔IgG (Rabbit IgG)为正常的兔IgG (Normal Rabbit IgG)，是一种未经任何标记的非特异性IgG (non-specific IgG)。本兔IgG磁珠作为对照磁珠时，可以排除IgG本身和特定目的蛋白或其它特定生物分子的非特异性结合。本产品进行免疫沉淀的流程参考图1。图1. 阿拉丁的UltraBio™ Rabbit IgG Magnetic Beads (兔IgG磁珠)免疫沉淀流程图。本产品非特异吸附低。与国内外大多数的同类产品相比，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0.5mg兔IgG抗体，该磁珠基本不会识别任何抗原。使用量参考正常抗体免疫磁珠的用量。本产品使用便捷。本产品储存在特殊保护液中，不含甘油，可以通过磁性吸附实现快速高效的分离，无需离心操作。本产品的主要指标如下表：4℃保存，两年有效。长期不使用，可以 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。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.兔IgG磁珠的准备。由于兔IgG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兔IgG磁珠，按照每500μl样品10μl或20μl磁珠悬浊液，取适量兔IgG磁珠至一洁净离心管中，加入1X TBS至最终体积为约0.5ml。说明：如果初始体积大于0.2ml，可以考虑先直接置于磁力架(FMS012/FMS024)上分离10秒，去除上清，然后再加入1X TBS 至最终体积为约0.5ml。b.用移液器轻轻吹打重悬兔IgG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 重悬兔IgG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兔IgG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兔IgG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3种方法之一进行洗脱。以下以兔源的Anti-Flag磁珠为例， 兔Anti-Flag磁珠采用一种方式洗脱时，兔IgG磁珠作为阴性对照，需要使用相同的洗脱方式。a.3X Flag竞争洗脱法：本方法为非变性法，洗脱效率高，且洗脱后的蛋白保持原有的生物活性，便于后续分析检测。(a)3X Flag多肽洗脱液的配制：取适量3X Flag多肽溶解于1X TBS中，使其终浓度为150μg/ml，或稀释5mg/ml的3X Flag多肽溶液至150μg/ml。(b)每10-20μl原始磁珠体积，加入100μl 3X Flag多肽洗脱液(150μg/ml)，混匀后置于侧摆摇床或旋转混合仪上，室温摇晃孵育30-60分钟，或4℃孵育1-2小时。为了提高洗脱效率，可延长孵育时间或重复洗脱。(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。上清即为洗脱的Flag标签蛋白。(d)洗脱的Flag标签蛋白置于4℃待用，或者-20℃或-80℃长期保存。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，并立刻加入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)洗脱并中和的目的蛋白置于4℃待用，或者-20℃或-80℃长期保存。注1：酸性洗脱法虽然高效，但仍可能低于竞争洗脱法或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)。c.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Flag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。 常见问题： Problem Possible Causes Solution Background is too high. Proteins bind nonspecifically to the rabbit IgG monoclonal antibody, insufficient washing on magnetic beads, or the microcentrifuge tubes. 1. Pre-clear lysate with Rabbit 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. Aladdin's UltraBio™ Rabbit IgG Magnetic Beads are based on amino magnetic beads, with 200nm particle size, covalently coupled with high-quality rabbit IgG antibody, generally used as the negative control of magnetic beads coupled with rabbit antibody against tags or target proteins in immunoprecipitation IP), co-immunoprecipitation (Co-IP), and chromatin immunoprecipitation (ChIP) assays.The rabbit IgG coupled with the Magnetic Beads is a normal, non-specific rabbit IgG without any labeling.UltraBio™ Rabbit IgG Magnetic Beads are used as the negative control for IP assays to address whether IgG itself is a source of non-specific binding. Please refer to Figure 1 for the workflow of IP assays with UltraBio™ Magnetic Beads.Figure 1. Workflow of IP assays with UltraBio™ Magnetic Beads.The UltraBio™ Rabbit IgG Magnetic Beads have lower non-specificity and smaller particular size, compared to most other similar products. Per milliliter of the magnetic bead suspension contains 10mg of magnetic beads coupled with no less than 0.5mg of rabbit IgG antibody.The UltraBio™ Rabbit IgG Magnetic Beads are convenient to use. Magnetic Beads are kept in a special protective solution without glycerol, and can be quickly and efficiently separated from the solution manually by using a magnetic stand or automatically by using an instrument.Main features of the UltraBio™ Rabbit IgG Magnetic Beads Precautions ： Repeated freezing and thawing of this product up to 3 times does not cause obvious changes in its performance.The pH of Rabbit IgG 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 cause the bead to aggregate. 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 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.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 appropriate lysis buffer. We recommend using Rabbit IgG Magnetic Beads used as the negative control are eluted in the same manner.(a) Preparation of 3X Flag elution buffer. Dissolve 3X Flag Peptide ( 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 Beads2mlP2151-1ml Streptavidin Magnetic Beads1mlP2151-200μl Streptavidin Magnetic Beads200μlP2151-5ml Streptavidin Magnetic Beads5mlP2171-1ml Mouse IgG Magnetic Beads1mlP2171-5ml Mouse IgG Magnetic Beads5ml