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

Thermo Scientific Pierce Direct Magnetic IP/Co-IP Kit uses NHS-chemistry to covalently immobilize IP antibodies onto magnetic beads for effective immunoprecipitation and co-immunoprecipitation.

Magnetic IP with NHS chemistry is convenient and allows for the immobilization of IP antibody from any species and subtype without the variations in coupling efficiency seen with Protein A and Protein G. The IP antibody is covalently attached so target proteins or co-IP protein complexes can be eluted and analyzed without antibody contamination. The kit contains an optimized protocol and all buffers and reagents necessary for antibody coupling and high yield IP or co-IP using either manual magnetic stands or automated platforms such as the Thermo Scientific KingFisher Instruments.

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

Antibody-free IP—antibody is irreversibly attached to the magnetic beads, resulting in negligible co-elution of intact antibody or heavy and light chains with the purified antigen
Fast—complete antibody conjugation and immunoprecipitation in less than 4 hours
Convenient—the IP/co-IP kit contains magnetic beads and all essential buffers for optimal immunoprecipitation
Antibody compatible—use with any antibody species, class or isotype
Low nonspecific binding—the bead surface is pre-blocked and all nonreacted NHS-ester groups are fully quenched
Protocol-compatible—protein coupling to the beads and immunoprecipitation can be performed manually or by automation (e.g., Thermo Scientific KingFisher Instruments)
Scalable—use only the amount of antibody needed for a single IP experiment or prepare a larger quantity of antibody magnetic beads for multiple experiments

The protocol for the Pierce Direct IP/Co-IP Kit conjugates IP antibody to Pierce NHS-Activated Magnetic Beads through NHS-ester chemistry. The antibody-bound beads are then incubated with quenching buffer to block remaining active NHS sites. The prepared antibody-bound beads are incubated with cell lysate or tissue extract that contain the protein antigen of interest, allowing the antigen:antibody complex to form. The beads are washed to remove nonbound material and a low-pH elution buffer is used to dissociate bound antigen from the antibody-bound bead. Neutralization buffer is included to prevent precipitation of the isolated antigen and to ensure protein activity in downstream applications. Lane Marker Sample Buffer is included for preparing samples for SDS-PAGE analysis. The protocol is optimized for 2 to 10μg of IP antibody per assay. For optimal co-IP yields, use 10μg of antibody. The beads can be processed manually using a magnetic stand or by automation with an instrument such as the Thermo Scientific KingFisher Flex.

Traditional IP with Protein A/G Magnetic Beads, involving passive, noncovalent coupling of antibody with Protein A/G typically provides higher antigen yields than IP protocols that involve covalent attachment of antibody to magnetic beads through crosslinking or NHS-ester chemistry. Covalent attachment inevitably causes loss of some functional antibody binding sites. To overcome this limitation with NHS-chemistry, it may be necessary to use larger amounts of IP antibody in the direct IP method than in the traditional IP method. The advantage of immunoprecipitation with IP antibody covalently bound to the magnetic bead is Western blots and gels devoid of interfering antibody bands.

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

Thermo Fisher Scientific Pierce GST Tag Protein Interaction Pull-Down Kits contains the necessary components to capture and purify proteins that interact with GST-tagged fusion proteins.

Features of GST Tag Protein Interaction Pull-Down Kit:

GST pull-down (Product No. 21516)—gently purifies protein interactors of any GST-tagged fusion protein without denaturing
Complete kits—provide all components and detailed protocol for purifying protein:protein interactions
No special equipment needed—use common laboratory equipment and reagents (e.g., microcentrifuge)
Convenient—microcentrifuge spin columns facilitate simple and efficient manipulation of agarose beads, including simple processing of multiple samples
Flexible—instructions include protocols for use with bait and prey proteins from many different sources
Binding—Binds approx. 8 mg of GST-tagged fusion protein per mL of resin

Applications:
• Discover a new protein:protein interaction from a cell lysate
• Confirm a putative interaction from a cell lysate or with a previously purified protein
• Extract protein:protein interaction information from in vitro transcription/translation lysates

You provide the tagged fusion protein as the "bait" and the cells expressing the putative protein interaction target ("prey"), and the Pull-Down Kits provide everything else: cell lysis buffer, microcentrifuge spin columns, tag-specific affinity resin (agarose beads) and optimized buffers and protocol. The Pull-Down Kits are designed to teach the method to first-time users and to increase ease-of-use, convenience and reproducibility for experienced researchers.

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Pierce™ HA-Tag IP/Co-IP Kit (Thermo Scientific™)

The Thermo Scientific Pierce HA Tag IP/Co-IP Kit provides the affinity resin, positive control and other reagents necessary to perform immunoprecipitation (IP) or co-immunoprecipitation (Co-IP) reactions with an HA-tagged bait protein.

Features of the HA-Tag IP/Co-IP Kit:

Improved—updated kit includes more immobilized antibody resin and improved protocol along with GST-PI3K-SH2-HA fusion protein as a positive control
Specific—highly specific anti-HA monoclonal antibody provides high-yield immunoprecipitation products and clean Western blot detection
Compatible—includes protocols and reagents for multiple elution conditions to accommodate different protein sensitivities and downstream applications
Robust—the kit and the anti-HA agarose are compatible with various cell lysates and physiologic (non-denaturing) buffer systems
Convenient and easy—complete kit includes all necessary reagents, convenient spin columns and easy-to-follow instructions

This kit is based on crosslinked beaded agarose to which a highly specific anti-HA antibody is covalently immobilized. The ready-to-use affinity resin, together with the included buffers, microcentrifuge spin columns, positive control, and easy-to-follow instructions, constitute a complete set of reagents sufficient for 25 for IP or Co-IP assays. Upon incubation with a sample containing the tagged fusion protein, interaction complexes involving the HA-tagged bait protein are captured on the agarose beads. After simple washing steps, the specific protein interaction complex is easily eluted from the resin in the supplied elution buffer or SDS-PAGE sample loading buffer for subsequent analysis.

The hemagglutinin (HA) peptide (YPYDVPDYA), derived from the human influenza virus HA protein is one of several fusion protein tags used for recombinant protein expression. Utilizing a specific, high-affinity immobilized antibody, HA-tagged fusion proteins can be quickly purified from bacterial and mammalian cell lysates as well as from the Pierce Human in vitro translation reactions. For co-immunoprecipitation reactions, simple wash steps allow enrichment and elution of specific protein interaction complexes into the supplied elution buffer or SDS-PAGE sample loading buffer for subsequent analysis.

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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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