货号：CB0234
存储条件：-20ºC保存，一年有效。Protein A/G Agarose Beads 4ºC保存（不可-20℃冻存）。
产品组分
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产品货号
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名称
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规格（20T）
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规格（100T）
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P0233
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Protein A/G agarose beads
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1ml
|
5*1ml
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C0101
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Protease Inhibitor Cocktail (100X)
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1ml
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5*1ml
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CB0234-01
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动物裂解缓冲液
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50ml
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250ml
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CB0234-02
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漂洗缓冲液
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50ml
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250ml
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CB0234-03
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洗脱缓冲液
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1ml
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5*1ml
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CB0234-04
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中和缓冲液
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1ml
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5*1ml
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9003-02
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Normal Rabbit lgG (1mg/mL)
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20ul
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100ul
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9012-01
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Normal Mouse lgG(1mg/mL)
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20ul
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100ul
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产品简介
本免疫沉淀试剂盒适合用于无标签蛋白、内源性蛋白等的下拉富集实验。试剂盒包含实验所需各种缓冲液，Protein A/G agarose beads 配合客户自配的特异性抗体便捷快速的完成免疫沉淀或者免疫共沉淀实验过程。本试剂盒适用于动物细胞样品的免疫沉淀过程，试剂盒中的缓冲液配方经过优化可以充分裂解细胞样本，有利于蛋白的释放和后续IP/Coip实验的进行。试剂盒提供的Normal Rabbit/mouse IgG可作为阴性对照排除beads的非特异性结合，如IP一抗为其他来源则需选择与之来源相同的IgG作为对照。
使用说明：
1. 蛋白提取
1.1 悬浮细胞样本
250-1000×g室温离心5min收集细胞。如必要可使用PBS洗涤一次，然后吸净残留的液体。轻轻vortex或者弹击管底以把细胞尽量分散开。可参考每5-10x105细胞加入100-200μl的比例加入的动物细胞裂解缓冲液（用之前按照1:100比例添加Protease Inhibitor Cocktail (100X)）。轻弹管底或适当吹打，以充分裂解细胞。充分裂解后应没有明显的细胞沉淀。如果细胞量较多，建议分装成5-10x105细胞/管，然后再裂解。大团的细胞较难裂解充分，而少量的细胞由于裂解液容易和细胞充分接触，相对比较容易裂解充分。充分裂解后，10,000-14,000×g在4ºC 离心3-5分钟，取上清，即可进行后续的免疫沉淀和免疫共沉淀等。
注：裂解后会出现少量不溶性物质，主要为基因组DNA等，离心后会产生沉淀物。
1.2 贴壁细胞样本
吸除培养液。如有必要，用PBS洗涤一次，然后吸净残留的液体。按照每5-10x105细胞(相当于6孔板的一个孔)加入100-200μl的动物细胞裂解缓冲液（用之前按照1:100比例添加Protease Inhibitor Cocktail (100X)），适当吹打，使裂解液和细胞充分接触。通常裂解液接触动物细胞1-2秒后，细胞就会被裂解。植物细胞宜在冰上裂解2-10min。充分裂解后，10,000-14,000×g在4ºC 离心3-5分钟，取上清，即可进行后续的免疫沉淀和免疫共沉淀等。注：裂解后会出现少量不溶性物质，主要为基因组DNA等，离心后会产生沉淀物。
1.3 检测蛋白提物中的蛋白浓度。并留存少量样本用于Input对照。
2免疫沉淀/共沉淀实验
2.1 琼脂糖珠清洗
琼脂糖珠存储在保护液中，使用前需要充分清洗。重悬混匀protein A/G琼脂糖珠后取适量混悬液（如总体积100ul，beads含量为25ul，吸取beads时建议用剪刀减去枪尖部分），添加漂洗缓冲液或植物裂解缓冲液至500ul，轻轻混悬吹打protein A/G琼脂糖珠，6000g 4℃离心30s，小心去除上清，不要吸到凝胶。重复以上步骤2-3次。
2.2 protein A/G 琼脂糖与抗体结合
（1）抗体稀释：按照抗体说明书的建议稀释抗体至工作浓度，或者配成终浓度为5-50ug/ml的抗体工作液，置于冰上备用。
（2）阴性对照设置：稀释试剂盒中带的与抗体来源相同的IgG，如一抗来源为Rabbit，则选Normal Rabbit IgG，稀释为与上两步抗体同样的浓度作为正常IgG工作液，用于阴性对照或者降低背景；
（3）抗体吸附：将2.1 步骤清洗完的protein A/G琼脂糖珠6000g 4℃离心30s，并吸除上清，加入100ul抗体工作液或IgG 工作液（目的抗体和IgG吸附可各做一组），重悬后室温在翻转混合仪上孵育15min-1h。
（4）洗涤：孵育完抗体后添加500ul 漂洗缓冲液，移液器轻轻吹打悬浮 protein A/G 琼脂糖珠，冰浴并置于摇床上5min，然后6000g 4℃ 离心30s，小心去除上清。重复此步骤3次。
（5）（可选）将洗涤后的protein A/G琼脂糖珠与样品在4℃孵育1小时后离心分离，以去除与IgG有非特异性结合的蛋白。
（6）取20ul吸附好抗体（目的抗体与IgG组各做一组）的protein A/G 琼脂糖珠添加到100ul蛋白样品中，置于摇床或者翻转混合仪上室温孵育2-4h或者4℃过夜孵育。孵育结束后，6000g 4℃ 离心30s，小心去除上清。可保留部分上清用于检测实验效果。
（7）洗涤：向（6）中去除上清的管子中添加500ul的漂洗缓冲液，吹打清洗琼脂糖珠，并重复清洗3-5次。此步骤同时可向漂洗缓冲液中按照1:100的比例添加C0101 蛋白酶抑制剂，以减少蛋白的降解。
2.3 洗脱
酸洗脱：向清洗干净的protein A/G 琼脂糖珠（20ul）中添加50ul 洗脱缓冲液，吹打混匀后在摇床或者翻转混匀仪上孵育5min，孵育时间不建议太长，最长不超过15min。
孵育结束后6000g 4℃ 离心30s，将上清转移到新的离心管中并添加5ul 中和缓冲液。为提高洗脱效率，洗脱步骤可重复1-2次。洗脱后的蛋白可在-20或-80长期保存，或者借用于后续检测分析。
变性洗脱：后续不用于其他检测分析的话，可使用变性洗脱的方式，在洗涤获得的protein A/G琼脂糖珠中添加50ul 2*loading buffer ，95℃加热5-10min。6000g 4℃ 离心30s，取上清可用于SDS-PAGE或WB检测。
注意事项
1. 本试剂盒中提供的洗脱方式为酸洗脱，如不需回收beads也可添加 loading buffer 进行煮样洗脱。
2. 实验过程中如涉及到磷酸化修饰或乙酰化蛋白，则需要添加磷酸酶抑制剂或去乙酰化抑制剂。
3. Beads使用前应保证充分混匀，混匀方式不建议剧烈涡旋震荡。
4. 本产品仅限专业人员科学研究使用，不可用于临床诊断或者治疗，不可存放于普通住宅内。
5. 为了您的安全健康，请穿戴一次性手套操作。
Product No.: CB0232
Storage Conditions: Store at -20ºC, valid for one year. Protein A/G Magnetic Beads can be stored at 4ºC.
Product Components
| Product No. |
Name |
Specification (20T) |
Specification (100T) |
| P1233 |
Protein A/G Magnetic beads |
1ml |
5×1ml |
| C0101 |
Protease Inhibitor Cocktail (100X) |
1ml |
5×1ml |
| CB0232-01 |
Plant Lysis Buffer |
50ml |
250ml |
| CB0232-02 |
Washing Buffer |
50ml |
250ml |
| CB0232-03 |
Elution Buffer |
1ml |
5×1ml |
| CB0232-04 |
Neutralization Buffer |
1ml |
5×1ml |
| 9003-02 |
Normal Rabbit IgG (1mg/mL) |
20ul |
100ul |
| 9012-01 |
Normal Mouse IgG (1mg/mL) |
20ul |
100ul |
Product Introduction
This immunoprecipitation kit is suitable for pull-down enrichment experiments of tag-free proteins, endogenous proteins, etc. The kit contains various buffers required for the experiment. Protein A/G magnetic beads, combined with customer-prepared specific antibodies, enable convenient and rapid completion of immunoprecipitation or co-immunoprecipitation experiments. This kit is applicable to the immunoprecipitation process of plant samples. The buffer formula in the kit has been optimized to fully lyse plant samples, facilitating protein release and subsequent IP/CoIP experiments. The provided Normal Rabbit/Mouse IgG can be used as negative controls to exclude non-specific binding of beads. If the primary antibody for IP is from other sources, IgG from the same source should be selected as the control. This kit can complete immunoprecipitation experiments with the help of a magnetic rack (recommended) or by centrifugation.
Instructions for Use:
- Protein Extraction
1.1 Take an appropriate amount of plant tissue samples (such as leaves) 0.5g-1g, place them in liquid nitrogen for freezing, then grind the frozen plant tissue samples into powder in a mortar, grinding as fully as possible to destroy the cell walls. Transfer the powder to a 15ml/50ml centrifuge tube, add 1-2ml of Plant Lysis Buffer (C0101 Protease Inhibitor Cocktail (100X) should be added at a ratio of 1:100) for lysis, and mix thoroughly with the lysate on a rotator at 4°C for 15min.
1.2 To improve lysis efficiency, 200ul of glass powder can be added, and the mixture can be fully shaken at 600-800g centrifugal force for 30min (the step of adding glass powder is optional). After lysis, centrifuge at 16,000g for 30min, aspirate the supernatant into a new centrifuge tube, and discard the precipitate.
1.3 Determine the protein concentration in the protein extract. Retain a portion of the sample as Input control.
- Immunoprecipitation/Co-immunoprecipitation Experiment
2.1 Magnetic Bead Washing
Magnetic beads are stored in a special protective solution and need to be fully washed before use to avoid affecting the experiment. After resuspending and mixing the magnetic beads, take an appropriate amount of magnetic bead suspension (the beads content in 100ul suspension is 25ul, and a total volume of 100ul suspension can be taken), add Washing Buffer or Plant Lysis Buffer to 500ul, separate the magnetic beads using a magnetic rack, and remove the supernatant. Add Washing Buffer or Plant Lysis Buffer to 500ul again, and repeat washing the beads 2-3 times.
2.2 Binding of Protein A/G Magnetic Beads to Antibody
(1) Antibody dilution: Dilute the antibody to the working concentration according to the antibody instructions, or prepare an antibody working solution with a final concentration of 5-50ug/ml.
(2) Negative control setup: Dilute the provided Normal Rabbit/Mouse IgG in the kit. If the primary antibody for IP is from Rabbit, select Normal Rabbit IgG; if it is from Mouse, select Normal Mouse IgG, and dilute to the same concentration as the antibody in the previous two steps as a normal IgG working solution, which is used as a negative control to exclude non-specific binding of beads. If the primary antibody for IP is from other sources, IgG from the same source should be selected as the control.
(3) Antibody adsorption: Remove the supernatant from the washed magnetic beads, add 500ul of antibody working solution or IgG working solution (groups for primary antibody and IgG can be set up respectively), resuspend, and incubate on a rotator at room temperature for 15min-1h.
(4) Washing: After antibody incubation, add 500ul of Washing Buffer, gently pipette to suspend the protein A/G magnetic beads, place on a magnetic rack for 10s, and remove the supernatant. Repeat this step 3 times.
(5) (Optional) Incubate the washed magnetic beads with the sample at 4°C for 1 hour to remove proteins that non-specifically bind to IgG.
(6) Add 25ul of protein A/G magnetic beads adsorbed with antibody (groups for primary antibody and IgG can be set up respectively) to 500ul of sample, and incubate on a shaker or rotator at room temperature for 2-4h or at 4°C overnight. After incubation, place on a magnetic rack, and remove the supernatant after 10s. A portion of the supernatant can be retained to detect the experimental effect.
(7) Washing: Add 500ul of Washing Buffer to the tube from which the supernatant was removed in step (6), pipette to wash the magnetic beads, and repeat washing 3-5 times. In this step, C0101 protease inhibitor can be added to the Washing Buffer at a ratio of 1:100 to reduce protein degradation.
2.3 Elution
Acid elution: Add 50ul of Elution Buffer to the cleaned magnetic beads (25ul), pipette to mix, and incubate on a shaker or rotator for 5min. It is not recommended to extend the incubation time. After incubation, place on a magnetic rack, collect the supernatant after 10s, and add 5ul of Neutralization Buffer. To improve elution efficiency, the elution step can be repeated 1-2 times. The eluted protein can be stored at -20 or -80 for a long time, or used for subsequent detection and analysis.
Denaturing elution: If it is not used for other detection and analysis, denaturing elution can be used. Add 50ul of 2×loading buffer to the washed magnetic beads, heat at 95°C for 5-10min. Place on a magnetic rack and take the supernatant for SDS-PAGE or WB detection.
