A Better GTPase Assay for Drug Development

Cover of October Issue of Assay and Drug Development Technologies featuring GTPase-Glo™ Assay.The path to drug development is strewn with obstacles: Identifying targets; configuring assays to help identify targets or drugs; uncovering the right compound to affect the selected target without off-target effects and screening multiple compounds to eliminate or identify potential drugs. Without the right tools, compounds or target, identifying potential disease therapies becomes nearly impossible.

When it comes to a drug target for cancer, the Ras protein family is at the top of the list because the proteins are expressed ubiquitiously and found mutated in many types of cancer. Because Ras proteins are involved in transducing signals from the surface of cells, many of the resulting mutations produce an activated Ras, inducing uncontrolled expression of the genes that Ras controls. Ras proteins are small GTPases (20–25kDa) that comprise a larger superfamily of proteins divided into five subfamilies: Ras, Rho, Rab, Arf, and Ran. These proteins control diverse cellular activities, including cellular differentiation, proliferation, cell division, nuclear import and export, and vesicle transport. GTPases are guanosine-nucleotide-binding proteins with affinity for GDP or GTP and are able to hydrolyze GTP. When bound to GTP, GTPases are active (turned on) and interact with downstream proteins in the signaling cascade. When GTPases are bound to GDP, the proteins are inactivated (turned off) and no longer transduce signals. Continue reading

Nucleic Acid Quantitation by UV Absorbance: Not for NGS

schematic diagram of UV-Vis Absorbance Method

For UV-Vis Spectrophotometry, light is split into its component wavelengths and directed through a solution. Molecules in the solution absorb specific wavelengths of light.

This is the second in a series of four blogs about Quantitation for NGS is written by guest blogger Adam Blatter, Product Specialist in Integrated Solutions at Promega.

Perhaps the most ubiquitous quantitation method is UV-spectrophotometry (also called absorbance spectroscopy). This technique takes advantage of the Beer-Lambert Law: an observation that many compounds absorb UV-Visible light at unique wavelengths, and that for a fixed path length the abosrbance of a solution is directly proportional to the concentration of the absorbing species. DNA, for example has a peak absorbance at 260nm (A260nm).

This method is user friendly, quick and easy. But, it has significant limitations, especially when quantitating samples for NGS applications.

  1. UV-absorbance lacks sensitivity. Even with a perfectly pure sample, the lowest detectable concentration of DNA is 2ng/µl. When you are performing NGS applications, often you are working with even lower concentrations of nucleic acid.
  2. Unfortunately, DNA is not the only molecule that absorbs light at 260nm. Many other organic compounds, including proteins, common contaminants left over from purification methods like phenols, and other nucleic acids (RNA, ssDNA, primers), also absorb at 260nm. Because these non-template compounds will contribute to the absorbance reading, they can cause gross overestimation of the the sample concentration.

These limitations and complicating factors can lead to insufficient input for library preparation or uneven coverage in multiplex pools as a result of poor normalization. In some cases, you would not be able to discern results from background; in others the run might fail completely. For these reasons, many NGS assay instruction manuals recommend recommend avoiding UV-absorbance-based quantitation.

When Every Step Counts: Quantitation for NGS

13170MA-800x277This series of blogs about Quantitation for NGS is written by guest blogger Adam Blatter, Product Specialist in Integrated Solutions at Promega.

As sequencing technology races toward ever cheaper, faster and more accurate ways to read entire genomes, we find ourselves able to study biological systems at a level never before possible. From basic science to translational research, massively parallel sequencing (also known as next-generation sequencing or NGS) has opened up new avenues of inquiry in genomics, oncology and ecology.

Many commercial sequencing platforms have been established (e.g., Illumina, IonTorrent, 454, PacBio), and new technologies are developed every day to enable new and unique applications. However, all of these platforms and technologies work off the same general principle: nucleic acid must be extracted from a sample, arranged into platform-specific library constructs, and loaded into the sequencer. Regardless of the sample type or the platform used, every step throughout this workflow is critical for successful results. An often overlooked part of the NGS workflow is sample quantitation. Here we are presenting the first in a series of four short blogs about the critical step of quantitation in NGS workflows.

Sample input is critical to NGS in terms of both quality and quantity. Knowing how much DNA you have, often in nanogram quantities, can make the difference between success and failure. There are several key points in the NGS workflow where sample quantitation is important before you can proceed:

  • After initial nucleic acid extraction from the sample matrix and before proceeding with library preparation
  • Post-library preparation when pooling barcoded libraries for multiplexing
  • Final pooled library quantitation immediately before loading for sequencing

There are several common methods for quantitating nucleic acids: UV-spectroscopy, Fluorescence spectoscopy, real-time quantitative PCR (qPCR). Because of inherent differences in sensitivity, specificity, time and cost, each of these techniques pose certain advantages and disadvantages with respect to the specific sample you are quantitating. Our next three blogs will discuss each of these methods against the backdrop of quantitating samples for NGS applications.

Will Warmer Weather Wake the Sleeping Giant (Viruses)?

Artist's conception of Mimivirus structure, the first of the giant viruses identified.

Artist’s conception of Mimivirus structure, the first of the giant viruses identified.

Following the discovery of Mimivirus (1) the first virus with a particles large enough to be visible under the light microscope, two additional “giant” viruses infecting Acanthamoeba have been discovered Pandoravirus (2) and Pithovirus sibericum (3), the latter from a 30,000 year old Siberian permafrost. A fourth type was recently isolated from the same sample of permafrost by Legendre et al, and named Mollivirus sibericum (4).

Mollivirus sibericum has an approximately spherical virion (0.6 µm diameter) with a 651kb GC-rich genome that encodes 523 proteins. To further characterize the virus the researchers performed transcromic- and proteomic-based time course experiments.

For the particle proteome and infectious cycle analysis, proteins were extracted and then run a 4–12% polyacrylamide gel, and trypsin digests were performed in-gel before nano LC-MS/MS analysis of the resulting peptides. Proteomic studies of the particle showed that it lacked an embarked transcription apparatus, but revealed an unusual presence of many ribosomal and ribosome-related proteins.

When the researchers explored the proteome during the course of an entire infectious cycle, the relative proportions of Mollivirus-, mitochondrion-, and Acanthamoeba encoded proteins were found to vary consistently with an infectious pattern that preserved the cellular host integrity as long as possible and with the release of newly formed virus particles through exocytosis.

In an interesting footnote, the authors of this study point out the fact that two different viruses retain their infectivity in prehistorical permafrost layers should be a concern in the context of global warming and the potential to expose humans to primeval viruses.


1. La Scola, B. et al.   (2003) A giant virus in amoebae. Science  299, 2033.
2. Philippe, N. et al. (2013) Pandoraviruses. Amoeba virus with genomes up to 2.5Mb reaching that of parasitic eukaryotes. Science 341,281–6.
3. Legendre, M. et al. (2014) Thirty thousand year old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology. Proc.Natl. Acad. Sci. 111, 4274–9.
4. Legendre, M. et al. (2015)  In depth study of Mollivirus sibercum, a new 30,000 year old giant virus infecting Acanthamoeba.  Proc. Natl. Acad. Sci. 112, E5327–35 (online).

How Fruit Flies (and maybe Pigeons?) Navigate; A New Report

A rock dove, similar in plumage to a pigeon.

A rock dove, similar in plumage to a pigeon.

Several years ago an intriguing story of successful navigation in complex situation, by pigeons, the birds most often compared to rats, caught my eye.

Our backyard once had a coop full of pigeons, so I’m not a total stranger to their navigation abilities (nor am I a pigeon expert). My favorites were the tumbling pigeons.

But it didn’t take much time researching that article from 2012, to learn that one of the more hotly debated how-do-they-do-it topics is animal navigation, in particular, the ability of pigeons to navigate back to home/point A when released at point B.

So when it appeared online today, in Nature Materials, the story “A Magnetic Protein Biocompass” caught my eye. Continue reading

There and Back Again, Part 1

In 2014, Promega created a special incentive to reward field science consultants who help the scientific community take advantage of the our on-site stocking program. The winners had to meet ambitious criteria to receive 2 round-trip tickets to anywhere in the world, a week of paid vacation and spending money. Our four winners from 2014 will share photos and stories about their journeys in a semi-regular Friday feature on the Promega Connections Blog.

Today’s travelogue comes to us from Mica Zaragoza, a senior client rep, who used his award to travel to Australia and New Zealand.

When initially introduced to the ambitious Helix award, I was amazed at the prospect of selecting anywhere in the world to travel, while blogging about my the adventures. Both humbled and amazed to receive this opportunity, my wife and I embarked on a journey across the Pacific.

Hyde Park in Sydney, Australia.

Hyde Park in Sydney, Australia.

Sydney, Australia

Departing our home in Chicago, my wife Crystal and I started our journey with a 5-hour trip to San Francisco for a layover before the 14 hour journey to Sydney. After jumping into the future (Thurs became Saturday), our first visit was to Central Sydney’s Hyde Park.

Taking jet lag into consideration, we decided to double-down by freshening up and dropping luggage to kick off our day at 7:30am. My first Australian purchase? Coffee! Continue reading

Customized Kinase Selectivity Profiling Just Got Easier

11296971-DC-CR-KinaseOff-target activities of target compounds can become costly if they aren’t discovered until late in the drug research and discovery process. Therefore, knowing the inhibitory profile of your test compounds across a broad collection of kinases as quickly as possible is highly desirable.

However, screening against many kinases at once requires a universal platform that is still sensitive enough to detect inhibitor activity and assess selectivity and potency on kinases of different classes. The luminescent ADP-Glo™ Kinase Assay is a universal platform that measures kinase activity by quantifying the amount of ADP produced during a kinase reaction.

We have used the ADP-Glo™ Chemistry to develop highly sensitive assays for more than 170 kinases across the human kinome and further enhanced the assays for ease-of-use by developing the Kinase Selectivity Profiling Systems. These systems provide an easy-to-use, reliable platform for kinase inhibitor profiling in house.

And even better, we now provide an online Kinase Profiling System Designer so that you can design a custom Kinase Selectivity Profiling System to fit your exact experimental needs. Simply drag and drop the combination of kinases you need to create an 8-kinase strip and submit your order. Continue reading

A Grateful Keynote Speaker, Not-So-Clever Criminals and Some World War I History: Highlights from the 26th International Symposium on Human Identification

nullThose of us lucky enough to attend the 26th International Symposium on Human Identification (ISHI) can agree that the meeting was a resounding success once again this year—plenty of outstanding workshops, presentations and posters, great networking and learning opportunities and, of course, fun with new and existing friends and colleagues.

Now that we’ve all had a chance to recover from all of the excitement, let’s recap some of the meeting highlights.

Continue reading

Big Data. Bigger Hope.

Research Cancer diagnosis from blood dropA paper published last week in Cancer Cell describes a new method for cancer detection from a simple blood sample. So far, one limitation of this type of non-invasive “liquid biopsy” for early detection of cancer has been the inability to identify the nature of the primary tumor. This new method, based on sequencing mRNA from platelets, overcomes this limitation in spectacular fashion—providing accurate identification of the primary tumor location in 71% of the samples tested.

Human blood platelets contain small amounts of mRNA. The RNA profile of “tumor-educated” platelets changes in response to tumor growth as the platelets take up mRNA from tumor cells. In this study, the authors sequenced the platelet mRNA of various cancer patients and healthy donors, and then searched for cancer-associated expression profiles. Continue reading

The Promise of miRNAs as Therapeutic Agents in Treating Disease

When researchers first identified a new family of seemingly non-functional “junk” RNA molecules, it’s unlikely they could have predicted the power and promise of these nucleic acids. The small, non-coding, single-stranded RNAs – typically 21-25 base pairs in length – were first discovered over 20 years ago in C. elegans, yet they were quickly found to be ubiquitous in species from worms to flies to plants to mammals. The role of these novel RNAs in the regulation of developmental pathways in worms, coupled with their prevalence, inspired researchers to better understand their significance.

We now know that miRNAs (for microRNAs) serve as post-transcriptional repressors of gene expression by targeting degradation of mRNA or interfering with mRNA translation. While small, each can have a big effect; a single miRNA can regulate dozens to hundreds of distinct target genes. They’ve been implicated in a variety of critical cellular processes such as differentiation, development, metabolism, signal transduction, apoptosis and proliferation.

Tissue-specific expression patterns revealed that specific miRNAs are enriched in mammalian tissues including adult brain, lung, spleen, liver, kidney and heart.  More compelling was the identification of abnormal miRNA expression in tumorigenic cell lines. It’s no wonder that this growing family quickly became ripe for exploration in disease development.

Basic research on miRNA is making its way into the clinic.

Research on miRNA is making its way into the clinic.

Within only a few years, a rapidly expanding body of research supported the theory that miRNA expression may indeed play a role in the development of human diseases including cardiovascular disease, cancer, diabetes, cystic fibrosis, and liver disease. Investigations into the expression of miRNAs in cardiovascular disease, in particular, have demonstrated not only their value as disease markers, but also how their dysregulation is linked to disease processes.

More recently a new possibility is being explored: can miRNA be manipulated to interfere with disease progression? Continue reading