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Pierce™ Biotinylated Protein Interaction Pull-Down Kit (Thermo Scientific™)

The Thermo Scientific Pierce Biotinylated Protein Interaction Pull-Down Kit contains the necessary components to capture and purify interactors of a biotin-labeled protein or ligand.

Features of the Biotinylated Protein Interaction Pull-Down Kit:

• Provides a complete, affordable and easy-to-use strategy for discoverying protein:protein interactions
• Uses common laboratory equipment and reagents (e.g., microcentrifuges, mini-gels, protein stains)
• Adaptable to single- or multiple-sample demands
• Flexible pull-down format uses spin cups for easy and efficient manipulation of streptavidin agarose beads

You provide the biotinylated protein as the "bait" (see

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Pierce™ Classic Magnetic IP/Co-IP Kit (Thermo Scientific™)

The Thermo Scientific Pierce Classic Magnetic IP/Co-IP Kit enables highly effective and efficient immunoprecipitation (IP) and co-immunoprecipitation (co-IP) of antigens and protein-complexes using less than 10 µg of antibody and a magnetic separator.

Features of the Classic Magnetic IP/Co-IP Kit:

Compatible—magnetic beads based on Protein A/G recombinant protein ensures compatibility with most primary antibodies, whether from mouse or rabbit
Fast—immunoprecipitate in as few as 30 minutes to reduce background and improve the capture of transient protein complexes
Clean—immobilize your antibody to prevent contamination in your eluate
Resistant—no leaching of Protein A/G in the presence of detergents, low pH buffers or common mass spectrometry solvents
Efficient—immunoprecipitate with half the recommended volume of magnetic particles compared to other magnetic beads
Convenient—the comprehensive Co-IP kit contains the magnetic beads and all essential buffers to complete immunoprecipitation experiments

This immunoprecipitation kit uses high-quality Thermo Scientific Pierce Protein A/G Magnetic Beads and optimized buffers and flexible protocol to accomplish high-yield IP or co-IP with manual or automated magnetic-separation tools. The optimized Lysis/Wash Buffer optimizes yield and efficient binding of antibody-antigen and co-IP interactions. The relatively gentle, low-pH Elution Buffer dissociates the bound immune complex from the Protein A/G. Alternatively, the included Lane Marker Sample Buffer provides rapid, denaturing elution for direct SDS-PAGE analysis of IP products.

Includes:
Pierce Protein A/G Magnetic Beads, lysis/wash buffer, elution buffer, neutralization buffer and sample loading buffer

Immunoprecipitation with magnetic particles is performed much like IP with beaded agarose, except that separations are performed using a magnet rather than by centrifugation. The specific antibody is first added to the sample to form an immune complex that is then bound to the magnetic beads. The complex is washed to remove non-bound material and a low-pH elution buffer dissociates the bound immune complex from the Protein A/G. Alternatively, the Lane Marker Sample Buffer is included for dissociation using denaturing conditions or for downstream sample prep for SDS-PAGE analysis. The kit includes Thermo Scientific Pierce Protein A/G Magnetic Beads for fast and convenient magnetic isolation of antigens and optimized buffers for high antigen yield. The beads are removed from the solution manually using a magnetic stand or by automation with an instrument such as the Thermo Scientific KingFisher Flex Instrument.

It is generally known that longer antibody-sample incubations times (2 hours to overnight) usually provide greater yields in IP assays. It is often assumed that this greater yield comes with increased background. Our researchers have found that overnight antibody-sample binding followed by immunoprecipitation with the Pierce Classic Magnetic IP/Co-IP Kit does tend to increase yield but does not increase background. Thus, we recommend that researchers perform this binding step for at least 1 hour whenever possible.

Protocol Summary:
• Incubate cell lysate with IP antibody for 1 to 2 hours at room temperature or overnight at 4ºC.
• Bind antigen-antibody complex to Protein A/G magnetic beads for 1 hour at room temperature.
• Wash beads twice with IP Lysis/Wash Buffer and once with purified water.
• Elute the antigen/antibody complex.

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Pierce™ Direct IP Kit (Thermo Scientific™)

The Thermo Scientific Pierce Direct IP Kit uses an activated resin to covalently immobilize IP antibodies (any species or class) on agarose beads without the aid of Protein A/G, enabling immunoprecipitation without antibody interference.

The primary benefits resulting from this method are the opportunity to use any species or subclass of purified antibody (not just types that bind to Protein A or G) and the ability to purify target protein without contamination by the antibody. The method also makes it possible to immunoprecipitate antigens from serum samples without co-purifying non-target immunoglobulins. Finally, the kit uses microcentrifuge spin cups to effectively wash and separate samples from the beaded agarose resin.

Features of Direct IP Kit:

Requires less than 10 µg of antibody—optimized procedure requires only 2 to 10 µg of antibody for a single IP experiment, no more than is required for traditional IP methods
Easy and efficient scalability—use only the amount of antibody needed for a single immunoprecipitation or immobilize 100 to 200 µg of antibody to prepare ready-made IP affinity resin for many experiments
Minimal antibody contamination—antibody is irreversibly attached to the agarose beads so that co-elution of heavy and light chains with the purified protein is minimized (i.e., less than 5%)
IP with any species and subclass of antibody—Use chicken IgY, human IgE, mouse IgM or any other purified protein
Prepared antibody affinity resin is reusable—because the antibody is covalently immobilized and not inactivated by the mild elution procedure, the resin often can be used several times
Convenient sample handling—spin columns (see dimensions) eliminate resin loss and provide for efficient separation of solutions
Complete kit—package includes cell lysis buffer and sufficient reagents and spin columns to perform at least 50 antibody immobilizations and IP experiments
Requires purified antibody—IP antibody solution must be free of BSA, gelatin or other stabilizer proteins; for easy removal of these proteins, see our Antibody Clean-up Kit (Part No. 44600)

Applications:
• Immunoprecipitation followed by electrophoresis and excision of the protein for mass spectrometry or sequencing identification analysis
• Immunoprecipitation and SDS-PAGE analysis of a target protein whose molecular weight is similar to the heavy or light chain antibody fragment
• Immunoprecipitation of target proteins for subsequent analysis by nondenaturing methods (i.e., methods not involving SDS-PAGE)
• Immunoprecipitation with antibodies that do not bind to Protein A, Protein G or Protein A/G
• Immunoprecipitation from serum and other sample that contain immunoglobulins

Direct immunoprecipitation in two easy steps:

Step 1: Attach antibody to agarose resin: The first step in the Direct IP Method is to prepare the IP antibody affinity resin. Pure antibody (see Highlights) is incubated in phosphate buffer with an appropriate volume of AminoLink Plus Coupling Resin, an aldehyde-activated beaded agarose resin. During the incubation, various lysine epsilon-amines on the surface of the large antibody molecule react with aldehyde groups on the resin to form Schiff-base bonds that are then stabilized to secondary amine bonds in the presence of cyanoborohydride. The immobilization reaction, known as reductive amination, is nondenaturing and typically results in greater than 85% antibody coupling efficiency with minimal loss of antigen-binding function.

Step 2: Perform immunoprecipitation experiments: Once prepared, the antibody resin can be used in several parallel aliquots or in total for IP experiments. Antibody resin is incubated with a sample that contains the protein antigen of interest, allowing the antibody:antigen complex to form. After washing to remove nonbound (presumably undesired) components of the sample, the antigen is recovered by dissociation from the antibody with elution buffer supplied in the kit. The entire procedure is performed in a microcentrifuge spin cup, allowing solutions to be fully separated from the agarose resin upon brief centrifugation. Only antigen is eluted by the procedure, enabling it to be identified and further analyzed without interference from antibody fragments. Furthermore, the antibody resin can be reused for additional rounds of immunoprecipitation.

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Pierce™ Crosslink IP Kit (Thermo Scientific™)

The Thermo Scientific Pierce Crosslink IP Kit adapts the traditional IP method to include reagents and protocol for crosslinking IP antibodies to Protein A/G agarose to enable antigen immunoprecipitation without antibody contamination.

The primary benefits resulting from these features are the ability to purify target protein without contamination by the antibody and the ability to more effectively wash and separate samples from the beaded agarose resin.

Features of the Crosslink IP Kit:

Eliminates antibody contamination—antibody is irreversibly attached to the agarose beads so that co-elution of heavy and light chains with the purified protein is minimized
Easy and efficient scalability—use only the amount of antibody needed for a single IP experiment or immobilize 100-200 µg of antibody to prepare ready-made IP affinity resin for many experiments
Assay reliability and sample handling—Pierce Spin Columns eliminate resin loss and provide for more efficient separation of solutions than traditional IP methods that use only microcentrifuge tubes
Prepared antibody affinity resin is reusable—because the antibody is covalently immobilized and usually is not inactivated by the mild elution procedure, the resin often can be used several times
Complete kits—package includes sufficient reagents and spin columns to perform at least 50 antibody immobilizations and IP experiments

Applications:
• Immunoprecipitation followed by electrophoresis and excision of the protein for mass spectrometry or sequencing identification analysis
• Immunoprecipitation and SDS-PAGE analysis of a target protein whose molecular weight is similar to the heavy or light chain antibody fragment
• Immunoprecipitation of target proteins for subsequent analysis by nondenaturing methods (i.e., methods not involving SDS-PAGE)

The Pierce Crosslink IP Kit method involves capturing the IP antibody to Protein A/G Agarose resin and covalently immobilizing it to the support by crosslinking with disuccinmidyl suberate (DSS). The antibody resin is then incubated with the sample that contains the protein antigen of interest, allowing the antibody:antigen complex to form. After washing to remove nonbound (presumably undesired) components of the sample, the antigen is recovered by dissociation from the antibody with elution buffer supplied in the kit. The entire procedure is performed in a microcentrifuge spin cup, allowing solutions to be fully separated from the agarose resin upon brief centrifugation. Only antigen is eluted by the procedure, enabling it to be identified and further analyzed without interference from antibody fragments. Furthermore, the antibody resin often can be reused for additional rounds of immunoprecipitation.

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