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- 详细信息
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- 文献和实验
- 技术资料
- 英文名：
Nuclear Complex Co-IP Kit
- 供应商：
研卉生物
- 规格：
50 rxns
FFPE福尔马林固定、石蜡包埋组织染色质免疫共沉淀试剂盒
Active motif公司基于在ChIP方面的专长，首次开发出商品化的可用于福尔马林固定、石蜡包埋组织样本的ChIP试剂盒用于二代测序。ChIP-IT® FFPE Chromatin Preparation Kit包含特殊试剂和操作指南，用于从组织切片中提取高质量的染色质。由于从FFPE样本中提取的染色质产量很低，必须使用ChIP-IT® FFPE染色质免疫共沉淀试剂盒，该试剂盒是唯一的可用于有限染色质转录因子或组蛋白富集的ChIP试剂盒，在保证最低背景信号的同时，能提供较高的灵敏度。
ChIP-IT® FFPE 的优势
● 可以从病例切片或者石蜡包埋组织块等起始样本中提取高质量的染色质
● 可以从ng级的FFPE染色质样本中高灵敏富集DNA
● 优化的试剂可以降低非特异结合造成的背景
● 和磁珠捕获的方法相比，离心过滤洗涤的方法更快更简单，并且可以更好的保持结果的一致性
● 严格验证的操作步骤，从正常或肿瘤组织FFPE切片中提取的染色质可用于qPCR和ChIP-Seq 分析
● 染色质制备试剂盒和ChIP试剂盒都包含对照，有利于结果的验证
图1 ChIP-IT® FFPE染色质免疫共沉淀试剂盒操作流程
使用ChIP-IT® FFPE Chromatin Preparation Kit (货号53030)从FFPE组织块或组织切片中制备染色质。加入特异的DNA结合蛋白抗体进行孵育，使用Protein G 琼脂糖珠捕获抗体-DNA结合蛋白/DNA复合物，经过重力过滤洗涤后，将复合物解交联，蛋白酶K消化结合蛋白，收集DNA。ChIP富集的DNA可用于二代测序或全基因组测序。
图2 qPCR分析使用ChIP-IT FFPE 试剂盒从正常人和肿瘤结肠组织中富集的DNA片段
使用ChIP-IT FFPE Chromatin Preparation试剂盒从保存10年的正常人和肿瘤结肠组织FFPE切片中制备染色质。取250ng正常人组织染色质和185ng肿瘤组织染色质，使用组蛋白H3K4me3抗体和转录因子YY1抗体，用ChIP-IT® FFPE试剂盒进行ChIP实验，富集的DNA使用ChIP-IT qPCR Analysis Kit（货号53029）进行分析。H3K4me3的结果显示在某些肿瘤中GAPDH的表达是上调的。数据表示每1000个检测细胞中所检测到富集结合情况，代表三次重复实验所得原始数据的均值。
由于PPFE样本的固定和保存条件差异很大，因此不是所有的PPFE样本都能获得高质量的染色质。因样本的限制，经常有必要从多张组织切片中制备染色质用于后续的ChIP实验。ChIP-IT® FFPE Chromatin Preparation试剂盒提供足够的试剂和详细的操作步骤，每次可从10张组织切片中至少制备出200ng用于ChIP实验的染色质。在制备过程中，每个PPFE样本的操作方法可能不一样，甚至因为固定方法的不同，即使相同的组织，在匀浆和超声的过程中也会遇到不同的困难。
图3 使用ChIP-IT® FFPE Chromatin Preparation Kit 从FFPE组织切片中制备染色质的操作流程
使用ChIP-IT® FFPE Chromatin Preparation Kit，从10-20 µm的FFPE组织块，或者未染色的5-10 µm的FFPE组织切片中提取染色质。每次可同时从多达10张切片中提取足量的染色质用于后继的ChIP实验。每张片子经过脱蜡和再水化处理，加入裂解缓冲液，经匀浆处理后超声破碎30秒，再用酶切混合物消化处理。离心收集裂解物混合到同一管里进行超声处理；离心，qPCR分析制备的染色质。
| issue type | Sample used per chromatin preparation |
| Human Colon | Tissue block – five 20 µm sections |
| Human Kidney | Tissue block – twenty-five 20 µm sections |
| Human Lung | Tissue block – two 20 µm sections |
| Rat whole brain | 5 slides – two 5 µm sections |
| Rat hippocampus | 25 slides – two 5 µm sections |
由于从PPFE样本中制备的染色质含量比较低，因此不推荐用凝胶电泳分析，而是用qPCR。ChIP-IT® FFPE Chromatin Preparation试剂盒包含DNA standards 和 PCR primers，可对从PPFE样本中制备的染色质进行qPCR定量分析，同时也可以验证是否为可溶性的染色质来用于下游的ChIP。如有必要，可对染色质进行重复的超声处理以增加其可溶性及产量。
使用ChIP-IT® FFPE Chromatin Preparation试剂盒从2组（A和B）保存10年的正常人肾组织（标记为N）和肿瘤组织（标记为T）切片中制备染色质。每个样本重复2次。使用阳性对照GAPDH-2引物和DNA Standards对可溶的和不可溶的染色质片段进行qPCR定量分析。上图显示仅从B组的肿瘤组织获得了较高产量的可溶性染色质。从B组正常组织以及A组正常组织和肿瘤组织中制备的不溶性沉淀，用ChIP buffer稀释到终体积1ml，进行第二次超声处理。取25ul的可溶性和非可溶性部分进行解交联，蛋白酶K消化处理，使用GAPDH-2引物和DNA Standards进行qPCR分析。结果表明可溶性的染色质含量仍然很低，但混合两次制备的可溶性染色质，含量足够用于ChIP分析。
参考文献
1.Weng, L. et al. (2010) J Pathol., 222: 41-51.
2.Gu, H.., et al. (2010) Nat. Methods, 7: 133-136.
3.Fanelli, M. et al.(2010) PNAS, 107(50): 21535-21540.
4.Fanelli, M. et al.(2011) Nat. Protocols, 6(12): 1905-1919.
产品订购信息
|
供应商
|
产品编号
|
产品名称
|
规格
|
|
Active Motif
|
53045
|
ChIP-IT® FFPE
|
16 rxns
|
|
Active Motif
|
53030
|
ChIP-IT® FFPE Chromatin Preparation Kit
|
5 rxns
|
|
Active Motif
|
53029
|
ChIP-IT® qPCR Analysis Kit
|
10 rxns
|
simplified co-immunoprecipitation of nuclear protein complexes
The Nuclear Complex Co-IP Kit simplifies co-immunoprecipitation of nuclear protein complexes by providing high-quality reagents for both nuclear extract preparation and immunoprecipitation. The kit's extraction process maintains protein complexes contained in the nucleus, specifically those previously bound to DNA, while the stringency of the Co-IP reagents can be easily adjusted so you can better study protein/protein interactions of varying strength.
Antibody binding beads are not included in the Nuclear Complex Co-IP kit and must be supplied by the user. Active Motif recommends our Protein G Agarose Columns for easy and efficient capture of the IP complex. The Protein G Agarose Columns contain pre-washed protein G agarose beads that have been specifically engineered to reduce non-specific binding. The beads are provided ready to use in filtration columns that streamline the antibody binding, wash and elution steps.
Contents & Storage
Each Nuclear Complex Co-IP Kit supplies sufficient reagents to perform 50 co-immunoprecipitation experiments and provides Hypotonic Buffer, PBS, Phosphatase Inhibitors, Protease Inhibitor Cocktail, PMSF, EDTA, Digestion Buffer, Enzymatic Shearing Cocktail, Detergent, IP High and IP Low Buffers, 5M NaCl, BSA and DTT. Reagent storage conditions vary from room temperature to -20°C. Please download a manual for complete kit specifications and storage conditions. All reagents are guaranteed stable for 6 months when stored properly.
Co-Immunoprecipitation (Co-IP) is a powerful method used to study protein/protein interactions. In Co-IP, one antibody is used to immunoprecipitate a target antigen and also co-precipitate any bound interacting proteins within a sample. This complex is then detected by Western blot using a second antibody targeted against one of the bound interacting proteins. However, traditional Co-IP methods are not optimal for studying DNA-binding protein complexes as these complexes are often disrupted during the extraction process. In addition, unstable protein complexes are frequently affected by the salt and detergent composition of the buffers used in the immunoprecipitation process. The Nuclear Complex Co-IP Kit extraction and immunoprecipitation reagents were optimized to help maintain nuclear protein complexes, providing you with the best results possible.
The Nuclear Complex Co-IP method
In the Nuclear Complex Co-IP Kit method, nuclear extracts are prepared by collecting cells in ice-cold PBS with Phosphatase Inhibitors. Then, the cells are resuspended in Hypotonic Buffer to swell the cell membrane and make it fragile. Addition of Detergent causes leakage of the cytoplasmic proteins into the supernatant. After collection of the cytoplasmic fraction, the nuclei are lysed and the nuclear proteins are recovered in a low-salt buffer in the presence of the Protease Inhibitor Cocktail and PMSF. This is followed by the addition of a proprietary Enzymatic Shearing Cocktail. The use of low-salt buffers protects protein complexes in the nucleus; DNA digestion allows a gentle release of undissociated protein complexes from the DNA.
After the protein complexes are collected, an immunoprecipitation reaction is carried out to detect the bound proteins. Two different immunoprecipitation buffers with either a low or high stringency starting composition are provided. In addition, detergent and salt are provided separately to enable you to vary the salt and detergent concentrations. The addition of salt and detergent is ideal for use with robust protein/protein interactions because such conditions reduce background. However, as unstable protein complexes may not withstand high stringencies, the kit format enables stringency to be modified as required by each complex.
Figure 1: Flow chart of the Co-Immunoprecipitation procedure used in the Nuclear Complex Co-IP Kit.
Nuclear extract is prepared using a low-salt buffer and enzymatic shearing, which protects nuclear protein complexes and releases them from the DNA. Immunoprecipitation is then carried out and the protein complex is washed using buffers that whose stringency can be adjusted by addition of salt and detergent, which helps maintain the complex integrity while eliminating non-specific proteins. Western blot is then performed using a 2nd antibody directed against a 2nd protein of interest.
Nuclear Complex Co-IP Kit advantages
- Simple and efficient
- Extraction procedure preserves nuclear protein complexes without disrupting protein/protein interactions
- Flexible immunoprecipitation reagents enable detection of protein/protein interactions of varying strength
- No need for creating fusion constructs or two-hybrid libraries
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文献和实验Protocol for Co-IP of different proteins with RFP-MeCP2 Preperation of cells (for p100): • 1st day split 293T EBNA cells in DMEM (high glucose + 10% FCS + 5mM glutamine + 5µg/ml gentamycine) • 2nd day cells
Dynabeads® Co-Immunoprecipitation Kit
Co-Immunoprecipitation Optimization below) from which the desired protein complex is isolated. The Extraction Buffer is made with 5 × IP supplied in the kit. The 5 × IP is supplied as 5 × concentrate and in sufficient quantity to allow for 40 coimmunoprecipitation
Southern Hybridization Experiment Kit
This kit contains materials for six groups to perform Southern transfer and hybridization analysis using the included lambda DNA samples and biotinylated probe. The intellectual objective of the experiment is to determine the region
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