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Gene Regulation

Nuclear Complex Co-IP Kit

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.

 
Name Format Cat No. Price  
Nuclear Complex Co-IP Kit 50 rxns 54001 $325 Buy Now
Protein G Agarose Columns 30 rxns 53039 $320 Buy Now
5 rxns 53037 $65 Buy Now

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.

 
Flow chart of the Co-Immunoprecipitation procedure used in the Nuclear Complex Co-IP Kit
 
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

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.