Pull-down assay is a simple and sensitive in vitro approach to studying protein-protein interactions (PPIs). In methodology, pull-down assay is similar to co-immunoprecipitation. In experiments, they both use an affinity ligand to capture interacting proteins. However, there is a difference between co-immunoprecipitation and the pull-down assay. The co-immunoprecipitation captures protein complexes by using immobilized antibodies, while the pull-down assay binds interacting proteins by using a purified and tagged protein, as “bait”.

Figure 1. Schematics of the peptide pull-down assay. BPTF is a protein that in humans is encoded by the BPTF gene. HP1 (Heterochromatin Protein 1), is an important marker molecule in epigenetic research. (Wysocka J., 2015)

Many proteins function in association with partner proteins or as components of large multi-protein complexes. Understanding these protein interactions is critical for our understanding of biological pathways and cellular function. The pull-down assay can be used to quantitatively detect both of these proteins and study the protein-protein interactions. 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 coupled 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 Services at Creative Proteomics

With our professional platform, Creative Proteomics can provide the pull-down assays to suit the specific needs of your project. Our buffers and analytical systems are optimized and can be also adjusted based your project needs as well.

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.

Table 1. Types of tag of the bait

Our Advantages

• Perfect experimental facilities. We have complete downstream supporting facilities, and quantitative analysis can be further carried out.
• Low background, no stray band. Using SDS-PAGE silver staining, the sensitivity is 100 times higher than that of Coomassie blue staining, and the electrophoresis results are clearer.
• Reliable experimental data. To eliminate the influence of random factors, we set up multiple control groups. And to ensure the validity of the experimental results, we repeat the experiment three times.

Applications

• 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.

Creative Proteomics can provide professional services for protein pull-down assay. We have most advanced instruments and technical service groups to perform the entire procedure with high-quality and high-efficiency. As every project has different requirements, please contact our specialists to discuss your specific needs.

References:
1. Jia S Z.; et al. Relative Quantification of Protein-Protein Interactions Using a Dual Luciferase Reporter Pull-Down Assay System. PLoS One. 2011; 6(10): e26414.
2. Wysocka J. Identifying novel proteins recognizing histone modifications using peptide pull-down assay. Methods. 2015; 40(4): 339–343.