Capture and purify interactors of a biotinylated "bait" protein.

The Pierce Biotinylated Protein Interaction Pull-Down Kit contains the necessary components to capture and purify interacting proteins involving a biotin-labeled protein. 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: 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. The procedure is conceptually simple (Figure 1).

Figure 1. 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).
Figure 2. 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 (Product # 21124). SuperSignal West Dura Chemiluminescent Substrate (Product # 34075) was added, and the blot was exposed to CL-XPosure X-ray Film (Product # 34080).

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

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

Related Products:
Biotinylation Kits and Reagents
Pierce Spin Cups and Columns
Co-Immunoprecipitation Kit
GST-Tag and His-Tag Protein Interaction Pull-Down Kits

References:

1. Jannatipour, M., et al. (2001). J. Biol. Chem. 276(35), 33093-33100.
2. Ducoux, M., et al. (2001). J. Biol. Chem. 276(52), 49258-29266.