Pierce Biotinylated Protein Interaction Pull-Down Kit
Capture and purify interactors of a biotinylated bait-protein.
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.
You provide the biotinylated protein as the "bait" (see Related Products for kits to biotinylate proteins) and the cells expressing the putative protein interaction target ("prey"). The Pull-Down Kit provides everything else you'll need: cell lysis buffer, microcentrifuge spin columns, streptavidin agarose beads and optimized buffers and protocol. The Pull-Down Kit is designed to teach the method to first-time users and to increase ease-of-use and convenience for experienced researchers.
Highlights:
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
Applications:
Discover a new protein interaction complex using a biotinylated protein as the bait to capture a putative binding partner from a cell lysate or other sample
Confirm expression of a known protein interaction target in a cell lysate
Determine the ability of two purified proteins to interact (bind) in various buffer conditions
Extract protein:protein interaction information from in vitro transcription/ translation lysates
Product Details:
Procedure summary for the Pierce Biotinylated Protein Interaction Pull-Down Kit. "Pull-down" is a small-scale affinity purification technique similar to immunoprecipitation (IP), except that the antibody function of is replaced by some other affinity system. In this case, the affinity system is the well-known and specific biotin-streptavidin interaction. The biotin-tagged protein acts as the "bait" to capture a putative binding partner (i.e., the "prey"). In a typical pull-down assay, the immobilized bait protein is incubated with a cell lysate. After the prescribed washing steps, the 'interactors" are selectively eluted for analysis in-gel or by Western blot. Because the biotin-streptavidin binding interaction is of such high affinity, it is generally possible to elute the prey protein without also co-eluting the biotin-labeled bait protein, which remains bound to the streptavidin agarose beads.
Lane 1: MW Markers
Lane 2: blank
Lane 3: Prey protein standard (pure BPTI protein); the lower band is the active monomer of BPTI.
Lane 4: blank
Lane 5: No-bait negative control; BPTI-containing lysate sample processed in pull-down, but without using any biotinylated aCT bait protein.
Lane 6: Negative control lysate (from cells not expressing the BPTI prey protein); detected upper band is an unknown cross-reacting protein produced by the mammalian cells.
Lane 7: Positive control: BPTI diluted in Tris buffer and processed through the pull-down procedure. Lower band is BPTI; upper band is unknown cross-reactor (see Lane 6).
Lanes 8-12: Eluted samples following different numbers of acetate buffer washes (decreasing from 5 washes in lane 8 to 1 wash in lane 12).
Biotinylated a -Chymotrypsin (baCT) used to pull-down its interactor bovine pancreas trypsin inhibitor (BPTI) from a mammalian cell lysate. Image is Western blot of eluates and controls following use of Pierce Biotinylated Protein Interaction Pull-Down Kit. Biotin a-Chymotrypsin (baCT) "bait" was bound to the streptavidin agarose beads. BPTI "prey" protein was expressed in and captured from a mammalian cell lysate. Upon elution of the BPTI, the baCT bait protein remained bound to the beads. Electrophoresis was by 4-20% Tris-Glycine SDS-PAGE. Following ransfer to nitrocellulose membrane, samples were probed with the Biotin-a-Chymotrypsin bait protein, then probed with Streptavidin-HRP (Part No. 21124). SuperSignal West Dura Chemiluminescent Substrate (Part No. 34075) was added, and the blot was exposed to CL-XPosure X-ray Film (Part No. 34080).
References:
Jannatipour, M., et al. (2001). J. Biol. Chem. 276(35), 33093-33100.
Ducoux, M., et al. (2001). J. Biol. Chem. 276(52), 49258-29266.
