Mutation Analysis Using HaloTag Fusion Proteins

In a recent reference, Kinoshita and colleagues characterized the phosphorylation dynamics of MEK1 in human cells by using the phosphate affinity electrophoresis technique, Phos-tag sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Phos-tag SDS-PAGE; 1). They found that multiple variants of MEK1 with diferent phosphorylation states are constitutively present in typical human cells.

To investigate the relationships between kinase activity and drug efficacy researchers from the same laboratory group conducted phosphorylation profling of various MEK1 mutants by using Phos-tag SDS- PAGE (2).

They introduced mutations in of the MEK-1 coding gene that are associated with spontaneous melanoma, lung cancer, gastric cancer, colon cancer and ovarian cancer were introduced into Flexi HaloTag clone pFN21AE0668, which is suitable for expression of N-terminal HaloTag-fused MEK1 in mammalian cells. Continue reading “Mutation Analysis Using HaloTag Fusion Proteins”

A BiT or BRET, Which is Better?

Now that Promega is expanding its offerings of options for examining live-cell protein interactions or quantitation at endogenous protein expression levels, we in Technical Services are getting the question about which option is better. The answer is, as with many assays… it depends! First let’s talk about what are the NanoBiT and NanoBRET technologies, and then we will provide some similarities and differences to help you choose the assay that best suits your individual needs. Continue reading “A BiT or BRET, Which is Better?”

Voted Drug Discovery and Development Product for 2019: NanoBRET TE Kinase Assays

Choice Drug Discovery and Development Product 2019 award
Michael Curtin, Promega, accepting the Reviewers’ Choice for Drug Discovery and Development Product of the Year award from SelectScience.

As announced at SLAS in Washington, D.C. recently, we are excited to have NanoBRET Target Engagement (TE) Intracellular Kinase Assays awarded the SelectScience Reviewers’ Choice for Drug Discovery and Development Product of the Year 2019!

The NanoBRET™ Target Engagement (TE) Kinase Assay, first available in the fall of 2017, has been getting great reviews on the SelectScience site for more than a year now. Continue reading “Voted Drug Discovery and Development Product for 2019: NanoBRET TE Kinase Assays”

It’s Almost iGEM Season—Help Is On The Way!

The 2019 iGEM Competition is on the horizon and team registration opens this month. We’re excited to partner with the iGEM Foundation again this year and offer our support to the young scientists who participate. If you’re starting an iGEM project, there are going to be things you need along the way. We are pleased to share a number of different ways we can help your iGEM team from now until the Giant Jamboree.

Grant Sponsorship

Tell us about your iGEM project and your team could win a 2019 Promega iGEM Grant Sponsorship. Ten winning teams will each receive $2000 in free Promega products to use for their iGEM projects. Tell us about your project—What problem are you addressing? What is your proposed solution? What challenges does your team face? Last year’s winning teams selected from a wide range of reagents and supplies, including master mix, restriction enzymes, ligase, DNA purification kits, expression systems, DNA ladders and markers, buffers and agarose. Click here to apply! Continue reading “It’s Almost iGEM Season—Help Is On The Way!”

Executing a NanoBRET™ Experiment: From Start to Data

This is a guest post from Katarzyna Dubiel, marketing intern in Cellular Analysis and Proteomics.

“The objective of my experiment was to test the NanoBRET™ assay as if I was a customer, independent of the research and development team which develops the assay.”

Designing and implementing a new assay can be a challenging process with many unexpected troubleshooting steps. We wanted to know what major snags a scientist new to the NanoBRET™ Assay would encounter. To determine this, we reached out to Laurence Delauriere, a senior applications scientist at Promega-France, who had never previously performed a NanoBRET™ assay. Laurence went step-by-step through the experimental process looking at the CRAF-BRAF interaction in multiple cell lines. In an interview, Laurence provided us with some tips and insights from her work implementing the new NanoBRET™ assay.

In a few words, can you explain NanoBRET?
“NanoBRET is used to monitor protein: protein interactions in live cells. It is a bioluminescence resonance energy transfer (BRET) based assay that uses NanoLuc® luciferase as the BRET energy donor and HaloTag® protein labeled with the HaloTag® NanoBRET™ 618 fluorescent ligand as the energy acceptor to measure the interaction of two binding partners.” Continue reading “Executing a NanoBRET™ Experiment: From Start to Data”

Optimizing Pressure Cycling Sample Preparation for Bottom-Up Proteomics

Large-scale analyses of the proteome have revealed proteomic changes in response to disease, and these changes hold great promise for diagnostics and treatment of complex disease if proteomic analysis can be brought into the clinical laboratory. Successful and reliable large-scale proteomics requires sample preparation workflows that are reproducible, reliable and show little variability. To bring proteomics into the clinical laboratory, standardized procedures and workflows for sample prep and analysis are required to generate valid, actionable results on a time scale useful for the clinic.

The two most common sample types analyzed for clinical proteomics are body fluids and tissue biopsies. To process these kinds of samples, there are two initial steps: tissue solubilization, followed by proteolytic digestion. Solubilization of solid tissues is the most labor-intensive and produces the most variable results.

The introduction of pressure cycling technology (PCT) using Barocycler instrumentation has greatly improved both tissue solubilization and digestion consistency. The PCT-based sample preparation protocols generally utilize urea as a lysis buffer for protein denaturing and solubilization. Urea has several drawbacks including inhibiting trypsin activity and introducing  unwanted modifications like carbamylation.

Lucas and colleagues analyzed whether replacing urea with SDC would produce similar tissue digestion profiles and improve the PCT method.

SDC allowed the use of higher temperatures compared to urea, and hence the first step (lysis, reduction, and alkylation) was performed at 56 °C. The second digestion step in the Barocycler was optimized, and the third step was eliminated. To further reduce digestion time, they capitalized on Rapid Trypsin/Lys-C.  Rapid Trypsin/Lys-C maintains robust activity at 70 °C, and allowed Barocycler digestion to be performed in a single step, completing digestion in 30 cycles (approximately 30 min) rather than 105 minutes, streamlining the protocol.

The data presented an improved conventional tissue PCT approach in a Barocycler by replacing urea and proteolytic enzymes with SDC, N-propanol, and modified commercially available enzymes that have higher optimum temperatures.

Paper Referenced

Lucas, N. et al. (2019) Accelerated Barocycler Lysis and Extraction Sample Preparation for Clinical Proteomics by Mass Spectrometry. J of Proteome Res 18, 399–405.

Deep in the Jungle Something Is Happening: DNA Sequencing

This blog was written by guest blogger and 2018 Promega Social Media Intern Logan Godfrey.

Only 30 years ago, the polymerase chain reaction (PCR) was used for the first time, allowing the exponential amplification of a specific DNA segment. A small amount of DNA could now be replicated until there was enough of it to study accurately, even allowing sequencing of the amplified DNA. This was a massive breakthrough that produced immediate effects in the fields of forensics and life science research. Since these technologies were first introduced however, the molecular biology research laboratory has been the sole domain of PCR and DNA sequencing.

While an amazing revolution, application of a technology such as DNA sequencing is limited by the size and cost of DNA sequencers, which in turn restricts accessibility. However, recent breakthroughs are allowing DNA sequencing to take place in jungles, the arctic, and even space—giving science the opportunity to reach further, faster than ever before. 

Gideon Erkenswick begins extractions on fecal samples collected from wild tamarins in 2017. Location: The GreenLab, Inkaterra.

Gideon Erkenswick begins extractions on fecal samples collected from wild tamarins in 2017. Location: The GreenLab, Inkaterra. Photo credit: Field Projects International.

The newfound accessibility of DNA sequencing means a marriage between fields of science that were previously largely unacquainted. The disciplines of genomics and wildlife biology/ecology have largely progressed independently. Wildlife biology is practiced in the field through observations and macro-level assessments, and genomics, largely, has developed in a lab setting. Leading the charge in the convergence of wildlife biology and genomics is Field Projects International. 

Continue reading “Deep in the Jungle Something Is Happening: DNA Sequencing”
Light enters eyes and is transmitted to SCN and PHb.

Light: A Happy Pill for Dark Days?

Have you ever had a day where you feel exceptionally good? As in take on the world kind of good? You feel so much better than the previous couple of days that you stop to wonder why.

Then it dawns on you.

The sun is out. It’s been cloudy for the past week but now—SUNSHINE.

You go out to lunch or for a walk just to take in those rays. Sure, it feels warmer than your darkened office space, but it’s the light rather than warmth that’s making a difference.

You purposely don’t wear sunglasses and it feels like the light is coming in through your eyes and massaging that part of your brain that is your happy zone. Are you imagining it or is the sun really affecting how you feel?

In a study reported in the September 2018 issue of Cell we learn that this is not a figment of your or my imagination (1). There is, in fact, a type of retinal cell that transports sunlight directly to the part of our brains that affects mood.

Eyes and the Body’s Master Clock

Circadian rhythms are innate time-keeping functions found in all multicellular organisms. This subject of the 2017 Nobel prize in Physiology or Medicine, circadian rhythms are fueled by daily light-dark cycles and are critical to the function of neurologic, immune, musculoskeletal and cardiac tissues (2). Nearly every mammalian cell is affected by circadian rhythms.

The human body has a circadian master clock, the suprachiasmatic nucleus or SCN. The SCN is a highly innervated tissue located in the hypothalamus (see image). It is connected directly to the retina by the optic nerve, and thus is influenced by external light and dark.

Light enters eyes and is transmitted to SCN and PHb.
Light enters the eyes and affects the SCN (physiologic effects), and as discussed in recent research, Fernandez et al. here, the perihabenular nucleus (behavioral effects). (Image in public domain.)

The retina of the eye is the light gathering instrument for this organ. Historically, it’s been understood that the retina is composed of two cell types, rods and cones, that function in transmitting light and images to the optic nerve, which sends those signals to the brain.

Drawing of the retina with rods and cones, some nervous tissues.
Some parts of the retina. Light enters the eye (from left) and passes through to the rods and cones. Here a chemical change converts the light to nerve signals. Image based on drawing by Ramón y Cajal, 1911 and licensed under Wikimedia commons.

Studies by Hattar et al. in the early 2000s identified that another cell found in the retina, the melanopsin-containing intrinsically photoactive retinal ganglion cells (ipRGCs) as the transmitter of circadian light signals (3). Through this direct connection to the SCN, the circadian master clock, the ipRGCs can influence a wide range of light-dependent functions independent of image processing (4).

Now Fernandez et al. have identified multiple types of ipRGCs. They showed that ipRGCs that mediate the effects of light on learning work via the SCN, while the pathway for light influencing emotions is different.

They discovered a new target of ipRGC cells, the perihabenular nucleus (PHb). The PHb is a newly recognized thalamic region of the brain. The authors showed that the connection between light and mood is regulated by ipRGCs through the PHb versus the SCN. They show that the PHb is integrated into other mood-regulating centers of the thalamic region.

You can see the details of their studies here.

In Conclusion

Daylight, and lack thereof, does affect both our mood and our ability to learn. In this 2018 report, we have learned that the pathways for these effects are distinct, and gain an understanding of a new thalamic region by which the light and mood actions occur. This information could influence development of better drugs and/or therapies for major depressive disorders.

For those of us with seasonal affective disorder, the evidence is undeniable—lack of light can cause issues, from sleep-wake problems, to mood and learning issues.

And while we can’t create sunshine, a special lamp or light box may help to gain some full spectrum light. To learn more about how to choose such a lamp and when to use it, see this Mayo clinic article for details.

References

  1. Fernandez, D.C. et al. (2018) Light affects mood and learning through distinct retinal pathways. Cell 175, 71–84.
  2. Ledford, H. and Callaway, E. (2017) Circadian clock scoops Nobel prize. Nature 550, 18.
  3. Hattar, S. et al. (2002) Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity. Science 295, 1065–70.
  4. Hattar, S. et al. (2003) Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice. Nature 424(6944)76–81.

How Prostate Cancer Cells Survive Glucose Deprivation

Illustration of energy metablism in cell.Glucose is an energy metabolite necessary for cellular survival and growth whether or not the cell is part of a tumor. Not only do cancer cells switch from oxidative phosphorylation to aerobic glycolysis (the Warburg effect) to gain more glucose, a hallmark of cancer, but they also increase the amount of glucose taken up from the surrounding extracellular space. However, the lack of glucose can have a negative effect on cells, causing them to become apoptotic in the absence of this metabolite. Cancer cells have methods to get around the requirement for glucose, including upregulating glucose transporters to improve access to the energy metabolite. In this Redox Biology article, researchers describe how activating androgen receptor in response to a lack of glucose affects the amount of GLUT1 expressed on prostate cancer cells, making the cells resistant to glucose deprivation.

To set the stage, two prostate cancer cell lines, LNCaP, an androgen-sensitive cell line, and LNCaP-R, an androgen-insensitive cell line, were deprived of glucose. Both cell lines showed signs of cell death, but LNCaP-R cells died in greater numbers. To probe how LNCaP cells died, several inhibitors (a pan-caspase inhibitor, two necroptosis inhibitors and a ferroptosis inhibitor) were added but did not change the way the cells died. However, an autophagy inhibitor enhanced cell death, suggesting the cells were necrotic not apoptotic. Teasing apart if the necrosis of LNCaP cells was due to glucose availability or merely disrupted glycolysis, the glucose analog 2DG was added to the medium with glucose. The cells survived when treated with 2DG, suggesting it was the absence of glucose that induced necrosis. When LNCaP cells were cultivated in medium that replaced glucose with mannose or fructose, the cells survived, another point in favor of sugar depletion causing cell death. Continue reading “How Prostate Cancer Cells Survive Glucose Deprivation”

Cell Free Application: Characterization of Long Non-coding RNA Inhibition of Transcription

Long noncoding RNAs have been shown to regulate chromatin states, transcriptional activity and post transcriptional activity (1). Only a few studies have observed long non-coding RNAs modulating the translational process (2). The noncoding RNA BC200 has been shown to inhibit translation by interacting with the translation initiation factors, eIF4A and eIF4B.

To characterize how BC200 translational inhibition could be controlled,  a variety of RNAs were transcribed/translated in vitro using the TNT system (Cat. #L4610) from Promega. To each transcription/translation reaction, BC900 RNA, hnRNPE1 and hnRNE2 proteins were added. Inhibition of BC200 activity was noted when proteins were successful expressed (3).

Literature Cited

  1.  Sosinska, P et.al. (2015) Intraperitoneal invasiveness of ovarian cancer from the cellular and molecular perspective. Ginekol. Pol. 86, 782–86.
  2. Geisler, S. and Coller, J. (2013) RNA in unexpected places: long non-coding RNA functions in diverse cellular contexts. Nat.Rev. Mol. Cell. Bio. 14,699–12.
  3. Jang, S. et. al. (2017) Regulation of BC200 RNA-mediated translation inhibition by hnRNP E1 and E2. FEBS Letters. 591, 393–5.