HaloTag Technology: a multifunctional technology enabling protein purification, analysis of protein interactions, cellular imaging, and surface display for overall study of protein function

In the post-genomic era a major objective is to fully characterize the human proteome involving identification of proteins, determination of their localization, modifications, interactions, activities and ultimately their function. Essential to this ambition is development of new affinity and imaging tools allowing comprehensive analysis of protein function. Here we present a new technology based upon the use of a protein fusion tag, termed HaloTag, which allows for highly specific, oriented, and covalent immobilization on surfaces, and also protein labeling based on covalent binding with a variety of fluorescent ligands. Because of the multifunctional nature of HaloTag technology, analysis of many different aspects of protein function can be successfully achieved using a single construct.


Abstract: "An introduction to HaloTag Technology and use for studying intracellular protein:DNA and protein:protein interactions"
This webinar will focus on the development of HaloTag, its ligands, and the variety of in vivo and in vitro applications designed for overall protein characterization. The Flexi vector cloning system, which allows for easy initial design and construction of HaloTag fusion vectors will also be discussed. In addition, data will be presented showing the ability to use HaloTag technology for study of intracellular protein interactions. Protein function is highly regulated through a complex network of protein interactions and modifications and to fully understand these processes, de-convolution of all types of protein interactions are necessary. To address these concerns, we have developed two novel methods; HaloCHIP and HaloTag pulldowns for the study in vivo protein:DNA and protein:protein interactions, respectively. HaloCHIP is an antibody-free alternative to the chromatin immunoprecipitation (ChIP) method wherein a DNA binding protein of interest is expressed as a HaloTag fusion, crosslinked, and directly captured on HaloLink resin. Similar to ChIP, crosslinks are reversed and captured DNA fragments are released for further analysis. Using the transcription factor CREB as a model system, HaloCHIP experiments coupled with DNA microarrays analysis were performed to analyze genome-wide CREB binding in HeLa cells. To study protein:protein interactions, traditionally a variety of methods have been employed including; yeast two hybrid screens, co-immunoprecipitation, and the use of affinity tags. We present here the use of the HaloTag fusion protein for isolation of intraceullar protein complexes from mammalian cells, including large macromolecular complexes such as the RNA Polymerases and the ribosome. The benefits of the rapid and covalent capture of HaloTag on its resin, allows for improved capture and success of complex isolation. The data shows compatible with a variety of downstream analyses including confirmation of known complexes using western blotting or identification of unknown binding partners using mass spectrometry. Combining each of these intracellular approaches with the other aspects of HaloTag technology, for example imaging and localization, allows for a more comprehensive understanding of protein function.

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