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Pierce Biotinylated Protein Interaction Pull-Down Kit


Capture and purify interactors of a biotinylated bait-protein.

Pierce Biotinylated Protein Interaction Pull-Down Kit

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 Pierce Biotinylated Protein Interaction Pull-Down Kit   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 αCT bait protein.
Example experimental results with Pierce Biotinylated Protein Interaction Pull-Down Kit
  • 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 α-Chymotrypsin (b-αCT) 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 α-Chymotrypsin (b-αCT) "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 b-αCT 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-α-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:

  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.

Related Resources:

Protein Interaction Pull-Down Kits FAQ

Related Products:

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


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
21115 Pierce Biotinylated Protein Interaction Pull-Down Kit
Formulation: Multi-component kit
Sufficient For: At least 25 pull-down reactions, each using 25µL of resin

Kit Contents:
Streptavidin Agarose Resin, 1.5mL
TBS Pack (makes 500mL), 1 pack
Biotin Blocking Solution, 15mL
Wash Buffer (pH 5.0), 100mL
Elution Buffer (pH 2.8), 50mL
Pierce Spin Columns and Accessories, 27 columns
Microcentrifuge Collection Tubes, 200 tubes
25-rxn kit Product Instructions for product #21115 Pierce Biotinylated Protein Interaction Pull-Down Kit MSDS for product #21115 Pierce Biotinylated Protein Interaction Pull-Down Kit Certificate of Analysis for product #21115 Pierce Biotinylated Protein Interaction Pull-Down Kit $409.00

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Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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