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

Catalog No. PI21115
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25 reactions kit
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PI21115 25 reactions kit
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Catalog No. PI21115 Supplier Thermo Scientific™ Supplier No. 21115
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Description

Easily capture and affinity-purify binding partners of any biotin-labeled protein or other ligand using these streptavidin agarose beads and complete reagent set.

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.

The kit makes it possible to perform an efficient pull-down assay with high protein recovery and reproducibility. This protocol uses immobilized streptavidin to capture a biotinylated protein and a spin column format that enables complete gel retention during each capture, wash and elution step. When the biotinylated bait protein is immobilized, it can withstand stringent washes and remains bound during prey protein elution, which results in an isolated prey protein without bait protein contamination. This technique is ideal when using native or recombinant proteins that are not expressed with a fusion tag. Additionally, prey protein is isolated from the immobilized bait protein using native binding conditions and nondenaturing elution conditions. The Biotinylated Protein Interaction 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:

  • Biotin pull-down – purifies protein interactors of any biotinylated protein or biotin-tagged ligand

  • Complete kit – provides all components and detailed protocol for purifying protein:protein interactions

  • No special equipment needed – uses common laboratory equipment and reagents (e.g., microcentrifuge)

  • Spin column efficiency – microcentrifuge spin columns facilitate microcentrifuge spin columns facilitate simple and efficient manipulation of streptavidin agarose beads, including simple processing of multiple samples

Includes:

Streptavidin agarose resin, TBS pack, biotin blocking buffer, wash buffer, elution buffer, spin columns and collection tubes

Recommended for:

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

Specifications

Content And Storage Sufficient For: At least 25 pull-down reactions, each using 25 μL of resin
• Streptavidin Agarose Resin, 1.5 mL
• TBS Pack (makes 500mL), 1 pack
• Biotin Blocking Solution, 15 mL
• Wash Buffer (pH 5.0), 100 mL
• Elution Buffer (pH 2.8), 50 mL
• Pierce Spin Columns and Accessories, 27 columns
• Microcentrifuge Collection Tubes, 200 tubes

Store at 4°C
Assay Affinity Purification
For Use With (Equipment) Microcentrifuge
Product Line Pierce
Quantity 25 reactions kit
For Use With (Application) Pull-Down (Biotin-Based)
Target Not Target-Specific
Type Pull-Down Kit
Sufficient For 25 Reactions
Format Kit

Frequently Asked Questions (FAQs)

If my protein is endogenously expressed, which Thermo Scientific Protein:Protein Interaction (PPI) kit should I choose?

We recommend the biotin kit for endogenously expressed proteins. The reason for this is the wide variety of biotinylation reagents that can be used to label the detection protein of the binding pair. For help choosing a biotinylation reagent, refer to the Biotinylation Selection Guide available at our website.
How is the tagged protein detected on the membrane using the Thermo Scientific Protein:Protein Interaction (PPI) kits?

The biotin kit utilizes streptavidin-HRP to detect the biotinylated protein bound to the prey protein on the membrane. The GST kit utilizes a polyclonal goat anti-GST antibody for detection of the glutathione-S-transferase tagged fusion protein bound to another protein on a membrane.
How many reactions can be performed using one Thermo Scientific Protein:Protein Interaction (PPI) kit?

The Thermo Scientific Pull-Down Protein:Protein Interaction Kits contain a complete, validated set of reagents specifically developed for performing pull-down assays.The kits contain sufficient materials for conducting 25 pull-down assays.
Are all of the conditions for binding the prey protein to the bait protein described in the Thermo Scientific Protein:Protein Interaction (PPI) kits?

No. Every protein pair will require unique conditions in order to bind with one another. Factors to consider are pH, ionic strength of the buffer, need for cofactors, whether the interaction is long term or transient, etc. The researcher is recommended to perform a literature search or try several different conditions in order to determine what conditions are best for the binding of their proteins of interest. The kit includes a “universal“ wash buffer formed by diluting the lysis buffer 1:1 in TBS.
With the Thermo Scientific Protein:Protein Interaction (PPI) kits, what methods can be used to detect the prey protein?

The prey protein can be detected following transfer on a membrane. The membrane procedures uses 1:100,000 - 1:500,000 dilution of the anti-GST antibody.
What types of Thermo Scientific Protein:Protein Interaction (PPI) kits are available?

There are Pull-Down Kits and Far-Western Kits. The Pull-Down Kits are very similar to an immunoprecipitation (IP), but instead of an antibody, a tagged bait protein [biotin (Cat. No. 21115) , GST (Cat. No. 21516) or 6xHis (Cat. No. 21277)] is used to bind the prey or target protein. This protein complex is then purified using an affinity column specific for the tagged bait protein. The Far Western Kits are similar to Western Blots, but again, instead of an antibody, a tagged non-immunoglobulin protein [biotin (Cat. No. 23500) or GST (Cat. No. 23505)] is used to bind to the prey or target protein for detection on the membrane.

For Research Use Only. Not for use in diagnostic procedures.