Rabbit IgG Agarose (兔IgG琼脂糖凝胶)

R749771-1ml

1ml

阿拉丁生产的Rabbit IgG Agarose (兔IgG琼脂糖凝胶)，也称Rabbit Normal IgG Agarose (兔正常IgG琼脂糖凝胶)、兔IgG IP Agarose (兔IgG 免疫沉淀琼脂糖凝胶)、Rabbit IgG Affinity Gel (兔IgG亲和凝胶)等，由高品质的未经任何标记的非特异性兔IgG (non-specific IgG)与琼脂糖共价偶联而成。本产品通常用作免疫沉淀(Immunoprecipitation, IP)、免疫共沉淀(Co-IP)、染色质免疫沉淀(Chromatin immunoprecipitation, ChIP)等免疫沉淀相关实验时兔来源抗体琼脂糖凝胶的阴性对照(Negative control)。本兔IgG琼脂糖凝胶中的兔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琼脂糖凝胶可以排除IgG本身和一些蛋白或其它生物分子的非特异性结合，从而用于降低背景或作为阴性对照。本产品非特异吸附低：本产品每毫升凝胶沉淀(settled gel)中IgG的含量高达约5mg IgG(通常每毫升凝胶沉淀中IgG的含量为约1-2mg)，用于特异性抗体在免疫沉淀之前的预处理去除非特异性结合(preclean)的效果会非常理想。同时，该琼脂糖凝胶基本不会识别任何抗原。使用量参考目的蛋白等分子免疫沉淀时抗体偶联琼脂糖凝胶的用量。本产品的主要指标如下表：本产品为50%凝胶悬液，包装体积为总体积，每毫升本产品中共含有0.5ml凝胶沉淀物。 注意事项 ： 本产品使用前一定要充分重悬，即充分颠倒若干次使混合均匀。本产品含有微量的防腐剂，不会影响常规的蛋白或蛋白复合物的纯化和免疫沉淀。但如果后续涉及酶活性测定，使用本产品前宜预先使用TBS等适当溶液洗涤凝胶3次，以充分消除防腐剂可能产生的干扰。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，或P1025/P1026蛋白酶抑制剂混合物(细菌抽提用)。高浓度的DTT、巯基乙醇、盐酸胍等对本产品的非特异性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。如果离心不能完全除去蛋白样品中的不溶物，可以将样品溶液用0.45μm的滤膜过滤。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。如下的样品制备步骤仅供参考，优先推荐使用特异性抗体偶联的琼脂糖凝胶进行免疫沉淀时的样品制备方法。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液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.Rabbit IgG Agarose (兔IgG琼脂糖凝胶)的准备。由于Rabbit IgG Agarose储存在含50%甘油以及防腐剂的保护液中，所以需要在加入样品前适当洗涤。a.轻轻重悬Rabbit IgG Agarose，尽量形成均匀的凝胶悬液，按照通常每100μl细胞裂解液中加入20μl混合均匀的凝胶悬液(以下免疫沉淀法中都以20μl凝胶悬液为例)，取适量Rabbit IgG Agarose至一洁净离心管中，加入适当的洗涤液如1X TBS 等至最终体积为约0.5ml。注1：使用大孔径吸头(如用剪刀剪去部分吸头)吸取凝胶悬液会比较方便。注2：此处及后续步骤中的1X TBS都优先推荐使用特异性抗体偶联的琼脂糖凝胶洗涤时的溶液，后续描述中的1X TBS仅作示例说明。b.轻轻重悬Rabbit IgG Agarose，6000×g在4℃离心30秒，小心去除上清，不要吸到凝胶。重复上述步骤两次。c.按照初始体积的量，用1X TBS 重悬Rabbit IgG Agarose。3.免疫沉淀(Immunoprecipitation, IP)。如下的免疫沉淀实验步骤仅供参考，优先推荐使用特异性抗体偶联的琼脂糖凝胶进行免疫沉淀时完全相同的实验步骤。a.加入凝胶与孵育。按照每100μl蛋白样品加入20μl凝胶悬液的比例加入经过洗涤的Rabbit IgG Agarose，置于侧摆摇床或旋转混合仪上，4℃孵育1-2小时。如需提高结合效率，可4℃孵育过夜。b.离心分离。孵育完毕后，6000×g在4℃离心30秒，小心去除上清，不要吸到凝胶。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，轻轻重悬Rabbit IgG Agarose，冰浴并置于摇床上5分钟，然后6000×g在4℃离心30秒，小心去除上清，不要吸到凝胶。重复洗涤三次。样品置于冰上用于洗脱。4.洗脱。如下的洗脱实验步骤仅供参考，优先推荐使用特异性抗体偶联的琼脂糖凝胶进行洗脱时完全相同的实验步骤。以下以Anti-Flag亲和凝胶和本产品为例进行说明。根据蛋白质特点及后续实验要求，可以选择下面3种洗脱方法之一。Anti-Flag亲和凝胶采用如下的一种方式洗脱时，兔IgG琼脂糖凝胶作为阴性对照，需要使用相同的洗脱方式。1)3X Flag竞争洗脱法：本方法为非变性法，洗脱效率高，且洗脱后的样品中不含Flag抗体的轻链和重链。a.3X Flag多肽储存液的配制：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20μl原始凝胶悬液体积，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。3X Flag多肽洗脱液体积一般为凝胶悬液的5倍。c.孵育完毕后，6000×g在4℃离心30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。2)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20μl原始凝胶悬液体积，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。酸性洗脱液体积一般为凝胶悬液的5倍。注：孵育时间不宜超过15分钟。c.孵育完毕后，6000×g在4℃离心30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Flag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)。3)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Western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P0015B SDS-PAGE蛋白上样缓冲液(2X)，但含有DTT等还原剂，用该上样缓冲液洗脱得到的样品中含有Flag抗体的轻链和重链。也可以自行参考《分子克隆》等配制不含DTT的2X SDS-PAGE蛋白上样缓冲液。b.每20μl原始凝胶悬液体积，加入20μl 2X SDS-PAGE上样缓冲液，95℃加热5分钟。c.6000×g在4℃或室温离心30秒，取上清进行SDS-PAGE电泳或WB检测。注：由于上样缓冲液中SDS会破坏Flag抗体，所以洗脱后的凝胶不能重复使用。常见问题： Problem Possible Causes Solution Large amount of tagged protein found in the flow through. Binding time is not enough. If using batch method, increase the binding time experimentally; If using column method, use a lower flow rate when loading samples. Column is overloaded. Reduce the amount of the sample added to the gel or increase the amount of gel. Tag is not accessible to gel. Expose the epitope tag by adding low amount of denaturant to the protein extract (dialysis may be needed before applying onto gel), or fuse thetag to the other terminus of the target protein. Gel has not been regenerated since last purification. Perform gel regeneration procedure prior to binding. Reagent compatibility problem. Dialyze the sample against TBS before purification procedure. The target protein has been degraded. 1. Prepare fresh lysates. Avoid using frozen lysates. 2. Perform purification at lower temperature, such as 4℃. 3. Use appropriate protease inhibitors in the lysate or increase their concentrations to prevent degradation of the fusion protein. Very few or notagged 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 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. 1. Reduce the number of washes. 2. Avoid adding high concentrations of NaCl to the mixture. 3. Use solutions that contain less or no detergent Incubation times are inadequate. Increase the incubation times with the affinity gel (from several hours to overnight). Interfering substance is present in sample. 1. Lysates containing high concentrations of DTT, 2-mercaptoethanol, or other reducing agents may destroy antibody function, and must be avoided. 2. Excessive detergent concentrations may interfere with the antibody-antigen interaction. Detergent levels in buffers may be reduced by dilution. 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 staining the membrane with Ponceau S. 3. Use fresh detection substrate or try a different detection system. 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. Background is too high. Proteins bind nonspecifically to the monoclonal antibody, the gel beads, or the microcentrifuge tubes. 1. Pre-clear lysate with Mouse IgG Agarose orRabbit IgG Agarose 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 from the lysate during the initial centrifugation of the affinity gel complexes. Aladdin's Rabbit IgG Agarose, also known as Rabbit Normal IgG Agarose, Rabbit IgG IP Agarose, or Rabbit IgG Affinity Gel, is covalently linked with the high-quality unlabeled non-specific rabbit IgG. This product is commonly used as a negative control for rabbit-derived antibody agarose gels such as Anti-Flag Affinity Gel in immunoprecipitation (IP), co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), and other immunoprecipitation-related assays.This product can also be used to reduce background by excluding non-specific binding of IgG itself to some proteins or other biological molecules.This product has high IgG binding capacity, with low non-specific adsorption. Per ml of settled gel contains approximately 5mg of IgG, which is ideal for removing non-specific binding. This product basically does not recognize any antigens. Main parameters of the Rabbit IgG Agarose Precautions ： This product must be fully resuspended by inverting the tube several times prior to use.This product contains a small amount of preservative, which does not affect routine IP assays and protein purification. But for special experiments that might be interfered by the preservative, the gel should be washed 3 times with appropriate solutions such as TBS prior to use.For immunoprecipitation assays, it is recommended to include both positive and negative controls. This product can be used for negative controls.Protein samples should be purified as soon as possible after collection and should always be placed at 4℃ or on ice to minimize protein degradation or denaturation. Protein degradation can also be inhibited by adding appropriate protease inhibitors, such as Protease Inhibitor Cocktail for General Use , Protease and Phosphatase Inhibitor Cocktail for General Use (MS-safe, 50X) , Protease Inhibitor Cocktail for Mammalian Cell and Tissue Extracts , Protease and Phosphatase Inhibitor Cocktail for Mammalian Cell and Tissue Extracts , etc.The non-specific binding capability of this product can be affected by high concentrations of DTT, mercaptoethanol, and guanidine hydrochloride, but it is applicable in Cell lysis buffer for Western and IP , RIPA Lysis Buffer , and NP-40 Lysis Buffer . For the main features and differences of various lysis buffers produced by and the selection of lysis buffers, please refer to the website at http://www.aladdin-e.com/support/lysis-buffer.htm.If the insoluble substance in protein samples can not be removed completely by centrifugation, samples can be filtered with a 0.45μm filter before performing the assay.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, such as ’s Cell Lysis Buffer for Western and IP . Under certain circumstances, ’s RIPA Strong Lysis Buffer , RIPA Medium Lysis Buffer , or RIPA Weak Lysis Buffer can be used. Other lysis buffers with a pH6-8 can also be used.a.After lysis and centrifuge, keep the supernatant at 4℃ or on ice for subsequent use. We recommend performing the subsequent procedures on the same day. Otherwise, make aliquots and store them at -80℃ for future use.2.Preparation of Rabbit IgG AgaroseAs the Rabbit IgG Agarose is stored in a protective solution containing 50% glycerol and a small amount of preservative, it needs to be washed properly before adding protein samples.a.Gently resuspend the Rabbit IgG Agarose, Transfer 10-20μl of gel suspension per 500μl of sample into a new centrifuge tube .Note: It is easier to aspirate the gel suspension using a wide orifice pipette tip (e.g., by cutting off part of the tip with scissors).b.Add 1X TBS (, ST661/ST665) to a final volume of approximately 0.5ml, and gently resuspend the Rabbit IgG Agarose by pipetting or vortex. Centrifuge at 6000×g for 30 seconds at 4℃, carefully remove and discard the supernatant. Repeat this step twice.c.Resuspend the Rabbit IgG Agarose with 1X TBS equal to the initial volume of gel suspension (e.g., if 10μl of gel suspension is taken, add 10μl of 1X TBS).3.Protein bindingd.Add 20μl of washed Rabbit IgG Agarose per 100μl of protein sample, mix well, and incubate at 4℃ for 1-2 hours or overnight at 4℃ with gentle shaking on a rotary mixer.e.After incubation, centrifuge at 6000×g for 30 seconds at 4℃, and carefully remove the supernatant.Note: A portion of supernatant can be reserved in a clean microfuge tube for examination of the binding results.f.Gently resuspend the Rabbit IgG Agarose in 500μl of 1X TBS and place in an ice bath on a shaker for 5 minutes. Centrifuge at 6000×g for 30 seconds at 4℃ and carefully remove the supernatant. Repeat the wash thrice, then keep the sample on ice for elution. 4.ElutionBased on the features of the target protein or further usage, one of the following three elution methods can be used. The following protocols are provided for eluting target proteins from the Anti-Flag Affinity Gel (, P2271). The Rabbit IgG Agarose negative control performed in parallel should be eluted in the same manner. 1)Elution with competitive peptide: This is a non-denaturing elution method with a high elution efficiency, and the eluted proteins do not contain the light and heavy chains of Flag antibody. a.Preparation of 3X Flag elution buffer: Dissolve the 3X Flag Peptide (, P9801-25mg) in 1X TBS buffer to a final concentration of 150μg/ml, or dilute the 3X Flag Stock Solution at 5mg/ml (, P9801) with 1X TBS buffer to a final concentration of 150μg/ml.b.For every 20μl of initial gel volume, add 100μl of 3X Flag Peptide elution buffer (150μg/ml). Resuspend the agarose gently and incubate for 30-60 minutes at room temperature on a rotary mixer , or 1-2 hours at 4℃. To improve the elution efficiency, increase the incubation time or repeat the elution. c.After incubation, centrifuge at 6000×g for 30 seconds at 4℃, and transfer the supernatant to a new microfuge tube. The supernatant contains the Flag-tagged protein and its protein complex.d.Store the supernatant at 4℃ for immediate use, or -20℃/ -80℃ for long-term storage.2)Acidic elution. This method is non-denaturing, relatively fast and efficient. The eluted proteins also retain their original biological activity, which facilitates subsequent analysis and detection.a.Preparation of acidic elution buffer (0.1M Glycine-HCl, pH3.0) and neutralization buffer (0.5M Tris-HCl, pH7.4, 1.5M NaCl). b.For every 20μl of initial gel volume, add 100μl of acidic elution buffer. Resuspend the agarose gently and incubate for 5 minutes at room temperature on a rotary mixer.Note: The incubation time should not exceed 15 minutes.c.After incubation, centrifuge at 6000×g for 30 seconds at 4℃, and transfer the supernatant to a new microfuge tube. Immediately add 10μl of neutralization buffer and mix well. d.To achieve the maximum elution efficiency, repeat steps b-c and combine the supernatant containing the Flag-tagged protein and its protein complex .e.Store the supernatant at 4℃ for or immediate use, or -20℃/-80℃ for long-term storage. Note 1: The elution efficiency of the acidic elution method might be lower than the other two elution methods.Note 2: The elution efficiency of the acidic elution method depends on characteristics of the target protein. To obtain a higher elution efficiency, the pH of the acidic elution buffer can be optimized from 2.5 to 3.1. The pH or amount of neutralization buffer needed to neutralize the eluates should also be adjusted appropriately.3)Elution with the 1X SDS-PAGE loading buffer. This is a denaturing elution method and the obtained protein samples are suitable for SDS-PAGE electrophoresis and Western blot analysis. a.Preparation of SDS-PAGE loading buffer: We recommend using ’s SDS-PAGE Sample Loading Buffer (2X) . Usually, the SDS-PAGE protein loading buffer contains a reducing agent such as DTT, and the eluted protein sample will contain the light chain and heavy chain of the Flag antibody.b.For every 20μl of initial gel volume, add 20μl of 2X SDS-PAGE sample loading buffer, mix well and heat at 95℃ for 5 minutes. c.Centrifuge at 6000×g or 4℃ for 30 seconds. Take the supernatant for SDS-PAGE electrophoresis or Western blot analysis.Note: The Agarose cannot be reused because SDS in the loading buffer destroys Flag antibodies.FAQ: Problem Possible Causes Solution Large amount of tagged protein found in the flow through. Binding time is not enough. If using batch method, increase the binding time experimentally; If using column method, use a lower flow rate when loading samples. Column is overloaded. Reduce the amount of the sample added to the gel or increase the amount of gel. Tag is not accessible to gel. Expose the epitope tag by adding low amount of denaturant to the protein extract (dialysis may be needed before applying onto gel), or fuse thetag to the other terminus of the target protein. Gel has not been regenerated since last purification. Perform gel regeneration procedure prior to binding. Reagent compatibility problem. Dialyze the sample against TBS before purification procedure. The target protein has been degraded. 1. Prepare fresh lysates. Avoid using frozen lysates. 2. Perform purification at lower temperature, such as 4℃. 3. Use appropriate protease inhibitors in the lysate or increase their concentrations to prevent degradation of the fusion protein. Very few or notagged 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 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. 1. Reduce the number of washes. 2. Avoid adding high concentrations of NaCl to the mixture. 3. Use solutions that contain less or no detergent Incubation times are inadequate. Increase the incubation times with the affinity gel (from several hours to overnight). Interfering substance is present in sample. 1. Lysates containing high concentrations of DTT, 2-mercaptoethanol, or other reducing agents may destroy antibody function, and must be avoided. 2. Excessive detergent concentrations may interfere with the antibody-antigen interaction. Detergent levels in buffers may be reduced by dilution. 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 staining the membrane with Ponceau S. 3. Use fresh detection substrate or try a different detection system. 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. Background is too high. Proteins bind nonspecifically to the monoclonal antibody, the gel beads, or the microcentrifuge tubes. 1. Pre-clear lysate with Mouse IgG Agarose orRabbit IgG Agarose 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 from the lysate during the initial centrifugation of the affinity gel complexes.