Typically protein analysis technologies and methods fall into two large buckets: the “biochemical” methods used for detection, purification and studying protein interactions, and the “cell-based” methods for understanding localization and trafficking. For the biochemical analyses, a researcher might employ tools like affinity tags or antibodies; for cell-based studies, a different set of antibodies or fluorescent proteins might be used. The end result is that to study how any one protein functions (where it is, what proteins it interacts with, when it is produced, when it migrates and is degraded) often requires several sets of clones to produce a variety of fusion proteins and a set of antibodies generated against a variety of epitopes.

An ideal protein analysis tool would be flexible enough to lead a researcher through the entire protein analysis workflow, allowing efficient capture and isolation, detection, real-time imaging with high signal and low background at all steps: one tag to find them all, one tag to bind them. In her Promega Webinar, “Accelerating Proteomics Research,” Jacqui Mendez introduced such a protein analysis tool.

The HaloTag® Technology uses a protein fusion tag the “HaloTag®” that is based on a small monomeric halophilic bacterial hydrolase. This protein has been engineered to covalently bind chloralkane substrates, resulting in an irreversible attachment. The binding is optimized for fast kinetics and stability, and because no homolog exists in mammalian cells, background is minimal.

With the HaloTag® Technology as the foundation, many tools have been created that establish an entire platform for virtually every step in understanding protein function from cloning to cellular localization to purification and identifying protein interactions. These tools include HaloTag® surfaces and resins for creating protein arrays, purifying proteins and identifying interacting proteins. The HaloTag® Fluorescent Ligands are used for cellular imaging, gel analysis and quantitation, and the HaloTag® Reactive Ligands allow custom modifications by the user.

For cloning, users have the choice of an extensive array of Flexi® Vectors designed for mammalian cell expression and also clones designed for bacterial expression of HaloTag® fusion proteins. These vectors allow directional cloning and simplified transfer (sequence only once and transfer) to other Flexi® Vectors.

Additionally, nearly 8,900 ORFs representing all major classes of proteins are available as N-terminal fusions into the pFN21A Flexi® Vector as part of collaboration between Promega and the Kazusa DNA Research Institute. All of these clones have been sequenced, insert size determined, and for most, expression has been validated by transfecting HEK-293 cells and analyzing with SDS PAGE. For nearly 80% of these clones, expression has also been verified by fluorescence microscopy. Determining whether your ORF of interest has been cloned and is available is as easy as searching www.promega.com/fmg with a name, keyword or sequence.

In short the HaloTag® Technology has provided a foundation that gives researchers a multi-functional handle on their protein of interest—tools that range from premade clones to ligands that allow real-time imaging to surfaces for efficient protein capture.

References

• HaloTag® Technology: Focus on Imaging Protocol
• HaloTag® Protein Purification System Protocol
• HaloTag® Mammalian Pull-Down and Labeling Systems Protocol
• HaloTag® Mammalian Protein Detection and Purification Systems Protocol
• HaloTag® Complete Pull-Down System Protocol
• HaloTag® Technology Overview

About the Webinar Series
www.promega.com/webinars provides a schedule of upcoming webinars. In addition there are links to previous webinars that allow you to either 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 not only view the webinar, but also to 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 links to recorded webinars.