Protein:Protein and Protein:DNA Interaction Analysis
Understanding which cellular molecules interact can help define protein pathways and recognition sites involved in gene expression. The HaloTag® Technology tool kit can capture both direct and indirect protein interactions as well as defining DNA binding sites and examining protein binding in live cells.
Characterize Binary and Higher Order Protein Complexes
Protein:protein interactions play critical roles in cellular processes, including replication, transcription, translation and signal transduction. The HaloTag® Mammalian Pull-Down Systems are designed to capture and purify intracellular binary and higher order protein complexes, including transient or weakly interacting partners. The covalent binding of the HaloTag® fusion bait protein means capturing the cellular protein complexes formed and identifying more physiologically relevant protein partners without interference from nonspecific binding.
To learn about identifying physiologically relevant protein interactions, this blog post describes how HaloTag® covalent labeling technology was used to understand ribosomal and GPCR interactions.
Overview of the HaloTag® Mammalian Pull-Down System
Capture Interacting Proteins for Investigation
Expected cytoplasmic binary and tertiary protein interactions in the NFκB pathway were identified with specific p65-HaloTag® pull-down. Proteins identified by mass spectrometry were RelA(p65), RelB, C-Rel, IκBa, IκBb, IκBe, p100, p105(p50) and p52.
Covalently Capture Protein:DNA Complexes without Antibodies
Protein:DNA interactions are integral to cellular processes such as transcriptional regulation and DNA modification. By identifying the DNA sequence that is the binding site for a protein or protein complex or by fishing potential protein binding partners from the cell, you are better able to determine what is a real interaction.
The HaloCHIP™ System is a novel method that covalently captures intracellular protein:DNA complexes without using antibodies and offers an efficient alternative to the standard chromatin immunoprecipitation (ChIP) method. Proteins of interest are expressed in cells as HaloTag® fusion proteins, crosslinked to DNA with formaldehyde and then captured on HaloLink™ Resin, which forms a highly specific, covalent interaction with the HaloTag® portion of the fusion protein. Stringent washing removes nonspecific proteins and DNA, and heating reverses the crosslinks between the DNA and the fusion protein and releases the captured DNA fragment, which subsequently can be purified.
Obtain data in 24–48 hours with fewer steps, minimizing potential experimental errors, and with improved signal-to-noise ratios, to detect small changes in protein-binding from a minimal number of cells. Plus HaloCHIP offers excellent reproducibility when compared to conventional antibody-depending ChIP assays.
Overview of the HaloCHIP™ System
Study Protein:Protein Interactions in Live Cells
Energy transferred from a NanoLuc® luciferase-Protein A fusion (energy donor) activates a fluorescently labeled HaloTag®-Protein B fusion (energy acceptor) when Protein A and Protein B interact.
Investigating dynamics of protein interactions can be challenging. Both induced and inhibited protein:protein interactions can be studied in real time under cellular conditions using the NanoBRET™ PPI Assay. Based on bioluminescence resonance energy transfer (BRET) technique where a bioluminescent donor protein brought into close proximity to a fluorescently tagged acceptor protein, the NanoBRET™ Assay harnesses the bright NanoLuc® luciferase as the energy donor and the HaloTag® protein labeled with a fluorophore as the energy acceptor. This sensitive PPI assay means you can study full-length proteins expressed at low levels.
Additional HaloTag® Resources
Covalently Capture Protein:DNA Complexes
This article describes mapping the intracellular interactions between protein and DNA—no antibodies needed.
Find Multiprotein Complexes in Cells
Learn about the localization of multiprotein Mediator complexes as well as isolating and characterizing their function in this poster.
Visualize Protein:Protein Interactions
Read this blog post to learn how the power of BRET can peer inside the cell to quantify, characterize and show proteins interacting.
Use a More Sensitive BRET Assay
See why the NanoBRET™ Assay is a great tool for shedding light on protein:protein interactions in this blog post.
Measuring Drug-Target Interactions
This webinar describes how the NanoBRET™ Assay can probe how possible modulators act on epigenetic proteins in live cells.