Top 5 Most Read Promega Papers in 2017

It’s always nice to know that someone is reading your paper. It’s a sign that your research is interesting, useful and actually has an impact on the scientific community. We were thrilled to learn that papers published by Promega scientists made the top 10 most read papers of 2017 in the journal ACS Chemical Biology. In fact, Promega scientists authored five of the top six most read papers! Let’s take a look at what they are.

#5 CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide

Publication Date (Web): September 11, 2017

This 2017 paper introduces our newest star: HiBiT, a tiny 11aa protein tag. To any scientist studying endogenous protein expression, the HiBiT Tagging System is your dream come true. It combines quantitative and highly sensitive luminescence-based measurement with a tiny-sized tag that can be easily inserted into endogenous protein via CRISPR/Cas9 gene editing with little impact on native protein function. The HiBiT Tagging System has been listed as a 2017 Top 10 Innovation by The Scientist, and it will drastically change how we study endogenous protein expression. Continue reading

Purify and Conjugate Antibodies in a Single Workflow

Isoform_Antibodies_LinkedInAntibodies labeled with small molecules such as fluorophore, biotin or drugs play a critical role in various areas of biological research,drug discovery and diagnostics. There are several limitations to current methods for labeling antibodies including the need for purified antibodies at high concentrations and multiple buffer exchange steps.

In a recent publication, a method (on-bead conjugation) is described that addresses these limitations by combining antibody purification and conjugation in a single workflow. This method uses high capacity-magnetic Protein A or Protein G beads to capture antibodies directly from cell media followed by conjugation with small molecules and elution of conjugated antibodies from the beads.

Using a variety of fluorophores the researchers show that the on-bead conjugation method is compatible with both thiol- and amine-based chemistry.

This method enables simple and rapid processing of multiple samples in parallel with high-efficiency antibody recovery. It is further shown that recovered antibodies are functional and compatible with downstream applications.

Literature Cited

Nidhi, N. et al. (2015) On-bead antibody-small molecule conjugation using high-capacity magnetic bead J. Immunol. Methods  http://dx.doi.org/10.1016/j.jim.2015.08.008

If We Could But Peek Inside the Cell …Quantifying, Characterizing and Visualizing Protein:Protein  Interactions

14231183 WB MS Protein Interactions Hero Image 600x214Robert Hooke first coined the term “cell” after observing  plant cell walls through a light microscope—little empty chambers, fixed in time and space. However,  cells are anything but fixed.

Cells are dynamic: continually responding to a shifting context of time, environment, and signals from within and without. Interactions between the macromolecules within cells, including proteins, are ever changing—with complexes forming, breaking up, and reforming in new ways. These interactions provide a temporal and special framework for the work of the cell, controlling gene expression, protein production, growth, cell division and cell death.

Visualizing and measuring these fluid interactions at the level of the cell without perturbing them is the goal of every cell biologist.

A recent article by Thomas Machleidt et al. published in ACS Chemical Biology, describes a new technology that brings us closer to being able to realize that goal. Continue reading

HaloTag® Research Application: Detection of Cancer Biomarkers

10242TAAntibodies labelled with radioisotopes or the sequential administrationof an antibody and a radioactive secondary agent facilitate the in vivo detection and/or characterisation of cancers by positron emission tomography (PET) or by single-photon emission computed tomography (SPECT) imaging.

There are drawbacks to both methods, including prolonged exposure to radiation and  ensuring that both the antibody and the radiolabelled secondary agent are suitably designed so that they bind rapidly upon contact at the tumor.

A recent publication (1) investigated a alternative method utilizing the HaloTag® dehalogenase enzyme HaloTag® is a dehalogenase enzyme (33 kDa) that contains an engineered cavity designed to accommodate the reactive chloroalkane group of a HaloTag® ligand (HTL). Upon entering the enzyme cavity, the terminal chlorine atom rapidly undergoes nucleophilic displacement, and a covalent adduct is formed, effectively anchoring the HaloTag® ligand in a precise location.

Three new HaloTag® ligands were synthesized and each labelled with the SPECT radionuclide indium-111  111In-HTL-1  and the dual-modality HaloTag® ligands,111In-HTL-2 and111;In-HTL-3 containing TMR which allows complementary imaging data).

For the validation of the pretargeting strategy based on these HaloTag® ligands, the target human epidermal growth factor receptor 2 (HER2)was selected. Trastuzumab (Herceptin®) was selected as the primary targeting agent and was modified with HaloTag® protein via the trans-cyclooctene/tetrazine ligation.

All three 111In-labelled HaloTa®g ligands exhibited significantly higher binding to the HER2 expressing when compared to negative controls.

Literature Cited

Knight, J. C et al.(2015) Development of an enzymatic pretargeting strategy for dual-modality imagingChem. Commun. 51, 4055–8.

Shedding Light on Protein:Protein Interactions with NanoBRET™ Technique

NanoBRET™ TechnologyIf you are trying to investigate protein:protein interactions inside cells, you know how important physiologically relevant results are. If you overload your cells with fusion constructs, your protein interactions may not actually reflect what is going on in the cell, and if your BRET energy donor and acceptor do not have sufficiently separated spectra, you can pick up a fair amount of noise in your experiment. Using the new superbright NanoLuc® Luciferase, and the HaloTag® Technology, we have developed a sensitive BRET system to help you take a better look specific protein interactions that interest you. Promega research scientist, Danette Daniels, describes the system in the Chalk Talk below:

Make and Use One Clone for Many Protein Analysis Applications

When I was characterizing proteins in graduate school, my life was filled with constructs, constructs, constructs. I made a variety of subclones to synthesize and isolate parts and pieces of the protein in vitro. I made clones and subclones to generate a panel of antibodies against different parts of the protein. Some of those antibodies ended up working best on Westerns; others performed better in immunocytochemistry experiments. There was no one tool or tag that could be used for every step in the characterization of the protein.

HaloTag® Technology: A single, multifunctional protein fusion tag.

HaloTag® Technology: A single, multifunctional protein fusion tag.

Halotag® fusion tag changes that.

The HaloTag® fusion tag spans both worlds of looking at proteins in isolation for studies of protein interactions and post-translational modifications to studying proteins in cells through real-time imaging or localization studies. Continue reading

Site-specific copy number variations in cancer: A story begins to unfold

Designed by Nick Klein for ISO-form, courtesy of Promega.

Designed by Nick Klein for ISO-form, courtesy of Promega.

Tumor cells are characterized by many features: including uncontrolled proliferation, to loss of contact inhibition, acquired chromosomal instability and gene copy number changes among them. Some of those copy number changes are site-specific, but very little is known about the mechanisms or proteins involved in creating site-specific copy number changes. In a recently published Cell paper, Black and colleagues, propose a mechanism for site-specific copy number variations involving histone methylation proteins and replication complexes.

Previous work from Klang et al. had shown that local amplification of chromosomal regions occurs during S phase and that chromatin structure plays a critical role in this amplification (2), and other work by Black and colleagues (3) implicated KDM4A in changing timing of replication by altering chromatin accessibility in specific regions. Other research also had shown that KDM4A protein levels influence replication initiation and that KDM4A has a role in some DNA damage response pathways (4,5).  Looking at the body of work, Black et al. hypothesized that KDM4A, with its roles in replication, might possibly provide link into the mechanism of site-specific copy number variation in cancer. Continue reading

How to Identify Physiologically Relevant Protein Interactions Using Covalent-Capture HaloTag(R) Technology Information

halotag_blogToday 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®. Continue reading

Another Step Closer to Understanding Epigenetic Gene Regulation

Chromatin fiberBack when I was a graduate student (more than a few years ago), I remember hearing another student joke that if a member of his thesis committee asked him to explain an unexpected or unusual result, he was going to “blame” epigenetics. At that time, the study of epigenetic gene regulation was in its infancy, and scientists had much to learn about this mysterious regulatory process. Fast forward to today, and you’ll find that scientists know a lot more about basic epigenetic mechanisms, although there is still plenty to learn as scientists discover that the topic is much more complicated than initially thought, as is often the case in science. A recent EMBO Journal article is contributing to our knowledge by shedding light on the role of the TET family of DNA-modifying enzymes in epigenetics (1).

Continue reading

One Tag to Rule Them All, One Tag to Find Them, One Tag to Bring Them All and in the Cell or Gel or Column Bind Them

Multiplex Live-Cell Imaging with HaloTag® protein

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. Continue reading