Today we can see inside the cell and identify protein interactions in their native environment. Many proteins have been characterized in a macromolecular complex, in an individual cell, or in the whole organism. We study proteins in their native environment because they rarely work in isolation. The study of intracellular protein interactions has been challenged by the ability to efficiently capture and preserve protein complexes, especially when attempting to isolate weak or transient interactions. In a recent webinar Rob Chumanov took us through techniques used to study proteins in their native environment and highlighted the most efficient method for studying them based on the HaloTag® covalent tag.
The older generation of protein tags is not ideal for studying protein interactions. These routine protein tags have been adapted for specific narrow applications, such as GFP for live-cell imaging and epitope tags (His, FLAG, and GST) for both fixed-cell imaging and capture of protein:protein interactions. As a consequence, often researchers create multiple protein fusion constructs with different tags in order to optimally characterize protein function. In contrast, HaloTag® technology provides broad flexibility for both imaging and biochemical applications with a single tag that binds rapidly, covalently, and specifically to synthetic small molecule ligands that ultimately determine the functionality of HaloTag®.
Applications with HaloTag® are endless. Examples of capturing small and large protein complexes are provided. Eukaryotic RNA polymerase complex associated proteins were captured by this technique. Rob has also presented the Ribosome story – showing how one of the most important macromolecular machines in the cell was characterized by placing the HaloTag® protein on the 40S subunit protein, Human RPS9. The most recent application of HaloTag® technology enabled better characterization of the Histone 1 (H1) interactome.
Besides protein interactions the HaloTag® technology enables real time monitoring of protein trafficking, pulse-chase, sub-cellular localization studies and other experiments. Red, blue or green fluorescent ligands are used to label proteins produced at different times so that one can watch their movement throughout the cell. The interchangeable labeling simplifies multiplexing with green fluorescent protein (GFP) or other labeling reagents, and the high sensitivity and low background levels make the HaloTag® platform an ideal tool for high-content assays. For example the imaging based GPCR internalization high content screening was developed with help of HaloTag® technology.
Finally Rob also explains the convenience of using premade ORF clones whose expression was already validated in HEK 293 cells. These clones were generated by the Kazusa Research Institute in Japan and turned into commercial products that shave off at least two months of cloning and validation time for those researchers wanting to study their protein of interest. Over >9,100 HaloTag®-ORF clones are available representing many gene families such as GPCRs, kinases, oncogenes and epigenetics-related genes.
HaloTag® technology is a comprehensive tool that bridges many gaps present in traditional protein research technologies with significant advantages in the study of protein interactions. Additional information regarding this new system can be found on the Promega web site.
About the Webinar Series
www.promega.com/webinars/ provides a schedule of upcoming webinars. In addition, there are links to previous webinars, which allow you to view the recording or download a pdf of the presentation. There is also a pdf of additional material available for each past webinar.
To register for a webinar, use the Registration link at: www.promega.com/webinars/ This allows you to view a live webinar and participate in the live chat.
Need a reminder? You can also sign-up for monthly invitations to webinars at the webinars page (see link above). Note: Live chat is only available for live webinars, not recorded webinars.
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