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

Catalog No. PI21277
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25 reactions kit
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PI21277 25 reactions kit
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Catalog No. PI21277 Supplier Thermo Scientific™ Supplier No. 21277
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Description

Easily capture and affinity purify binding partners of His-tagged fusion proteins using nickel-chelate (Ni-IDA) agarose beads and this complete reagent set.

Thermo Scientific™ Pierce PolyHis Protein Interaction Pull-Down Kit purifies protein interaction complexes involving practically any user-supplied His-tagged fusion protein.

The PolyHis Protein Interaction Pull-Down Kit contains the necessary components to capture and purify proteins that interact with recombinant proteins containing a histidine (6xHis) fusion tag. You provide the tagged fusion protein (bait) and cells expressing the putative protein interaction target (prey), and the pull-down kit supplies everything else: cell lysis buffer, microcentrifuge spin columns, cobalt-IMAC affinity resin (agarose beads), optimized binding and elution buffers, and a detailed protocol. This kit is designed to teach the method to first-time users and to increase ease-of-use, convenience and reproducibility for experienced researchers.

Highlights:

  • 6xHis pull-down – purifies protein interactors of any His-tagged fusion protein

  • 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)

  • Convenient – microcentrifuge spin columns facilitate simple and efficient manipulation of cobalt IMAC agarose beads, including simple processing of multiple samples

  • Flexible – instructions include protocols for bait and prey proteins from different sources

Includes:

Cobalt chelate agarose resin, lysis buffer, TBS pack, imidazole solution for elution, microcentrifuge spin columns and collection tubes

Recommended for:

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

Specifications

Content And Storage Sufficient For: At least 25 pull-down reactions, each using 25 μL of resin
• Cobalt Chelate Agarose Resin, 750 μL
• Pull-Down Lysis Buffer, 250 mL
• Imidazole Solution (4M), 5 mL
• TBS Pack (makes 500 mL), 1 pack
• Pierce Spin Columns and Accessories, 27 columns
• Microcentrifuge Collection Tubes, 100 tubes

Upon receipt store Pierce Lysis Buffer at room temperature. Store remaining kit components at 4°C.
Assay Affinity Purification
For Use With (Equipment) Microcentrifuge
Product Line Pierce
Quantity 25 reactions kit
For Use With (Application) Pull-Down (Tag-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.