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HaloTag® Technology for Protein:Protein Interactions

Protein interactions are critical to the majority of cellular processes, including: replication, transcription, translation and signal transduction. HaloTag technology provides a convenient method for characterization of protein interactions. 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.

  • Identify more physiologically relevant protein partners
  • Rapidly capture binary and higher order complexes directly from lysates
  • Transition from pull-downs into imaging studies using the same construct

HaloTag® Mammalian Pull-Down System Protocol Overview

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Study Simple Binary and Tertiary Interactions

In the HaloTag® Mammalian Pull-Down System, the covalent binding of HaloTag®-fusion proteins affords efficient protein recovery, and low non-specific binding.

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Expected cytoplasmic binary and tertiary protein interactions in the NFkB pathway were identified with specific p65-HaloTag® pull-down. Proteins identified by MS were RelA(p65), RelB, C-Rel, IkBa, IkBb, IkBe, p100, p105(p50), p52.
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Expected cytoplasmic binary and tertiary protein interactions in the NFkB pathway were identified with specific p65-HaloTag® pull-down. Proteins identified by MS were RelA(p65), RelB, C-Rel, IkBa, IkBb, IkBe, p100, p105(p50), p52.

Study Macromolecular Complexes

The HaloTag® Mammalian Pull-Down System gives consistent results and clean controls when pulling down macro-molecular complexes.

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The majority (26/29) of known subunits for the transcriptional coactivator complex, Mediator, were specifically isolated in a single pull-down experiment using MED26 as bait. The HT control demonstrates the low non-specific binding of this fusion tag in pull-downs since 0/29 subunits of Mediator were identified.
9712MA_1320x2240
The majority (26/29) of known subunits for the transcriptional coactivator complex, Mediator, were specifically isolated in a single pull-down experiment using MED26 as bait. The HT control demonstrates the low non-specific binding of this fusion tag in pull-downs since 0/29 subunits of Mediator were identified.

Easily Transition from Pull-Downs to Imaging Applications

With the HaloTag® technology, you can seamlessly transition from pull-down studies to imaging applications without additional cloning as all HaloTag®  Ligands are interchangeable. In the example below, U2OS cells stably expressing p65-HaloTag labeled with HaloTag TMRDirect™ Ligand show proper cytoplasmic localization.

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Live-cell imaging of p65-HaloTag® protein. U2OS cells stably expressing the p65-HaloTag® fusion protein were labeled with HaloTag® TMRDirect™ Ligand as described in the HaloTag® Mammalian Pull-Down and Labeling Systems Technical Manual #TM342 and imaged by confocal microscopy using appropriate filter sets. Panel A. Fluorescence channel. Panel B. DIC channel. Scale bar represents 20μm.
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Live-cell imaging of p65-HaloTag® protein. U2OS cells stably expressing the p65-HaloTag® fusion protein were labeled with HaloTag® TMRDirect™ Ligand as described in the HaloTag® Mammalian Pull-Down and Labeling Systems Technical Manual #TM342 and imaged by confocal microscopy using appropriate filter sets. Panel A. Fluorescence channel. Panel B. DIC channel. Scale bar represents 20μm.

HaloTag® Pull-Down System in the Literature

"Examining the Complexity of Human RNA Polymerase Complexes..."
J Proteome Res., 2012 Jan. 3 (2013).
Read article