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Pull-Down Assay

Introduction

Pull-down assay is a in vitro method used to concentrate the target protein or direct protein-protein interaction. It is often used by biochemists to verified the protein-protein interaction predicted by other assay. It utilize beads conjugated with bait protein, which is predicted to interact with the target protein, to trap the prey, followed by downstream analysis of bait-prey complex, for instance, Western blotting and mass spectrometry.

The principle of pull-down assay is affinity purification, which is similar to immunoprecipitation, except that this method uses bait protein, instead of antibody to trap the target protein. In a pull-down assay, a bait protein is tagged and immobilized on beads with affinity ligand specific for the tag, thereby generating a "secondary affinity support" for purifying other proteins that interact with the bait protein. When incubated with a protein sample that contains putative "prey" proteins, such as a cell lysate, the bait can bind the prey, and thus trap down the prey from the lysate.

In addition to investigating the interaction of two proteins, pull-down assays can also be used to detect the activation status of specific proteins. Activation of proteins are often couple with protein conformational change, and the bait selectively binds to a specific conformation. In this case, pull-down assay can be utilized to selectively purify the active prey. For instance, GTPase effectors only recognize the active GTPases, thus when using GTPase effectors as the bait, only the active GTPases are trapped and purified, and the proportion of active GTPase can thus be measured.

Pull-Down Assay

Type of tag of the bait

There are many types of tags for the bait of pull-down assay, such as GST, poly-His, and biotin, etc. The affinity ligand used to immobilize bait are glutathione, nickel and chelate complexes, and streptavidin, respectively.

Bait tag Affinity ligand
Glutathione S-transferase (GST) Glutathione
Poly-histidine Nickel or cobalt chelate complexes
Biotin Streptavidin

Creative Proteomics provides the above pull-down assays to suits the specific needs of your project. Our buffers and analytical systems are optimized and can be also adjusted based your project needs as well. You welcome to send us inquiry to discuss the specific needs of your project.

Features of Pull-Down Assay

  • Study the direct interactions between proteins and protein complex
  • Verify predicted protein-protein interaction
  • Analyze and quantify the specific isoforms of or active/inactive protein in the tested samples

Our Pull-Down Assay service contains:

  • Experiment design based on your specific project needs: buffers, beads, tag of bait, etc.
  • Parameters optimization, such as binding time
  • Cell lysis, pull-down assay: binding, washing, and elution
  • WesternBlot/ mass spectrometry, depending on the project needs
  • Data analysis & report delivery

Ordering Procedure:

Ordering Procedure


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