Cell Biology

What Can We Do to Make Your Work Easier? Addressing the Challenges of Working with Primary Antibodies

Posted on by Michele Arduengo, PhD

Scientific progress is iterative. Small advances and bits and pieces of knowledge are accumulated over time with each advance depending on the body of work that has preceded it. Being able to replicate the work from one step to another in this march of science is critical, and it is not easy. We all recognize that a well-documented procedure goes a long way toward enabling reproducibility, but the procedure is not the only hurdle to reproducibility, and it’s not even the main one.

Artistic Image of Hibit Tag

Many of the reagents researchers use are derived from or produced by living organisms. A decade ago, in 2015, an analysis of preclinical research estimated that irreproducibility costs the U.S. around $28 billion a year. When the authors traced the causes, the largest single category wasn’t study design or data analysis. It was biological reagents and reference materials (1).

When Bill Linton first developed the idea of a company that supports scientists in the 1970s, his question was: “What can we do to make your work easier?” The answer he kept getting was the same: researchers wished they could buy reliable restriction enzymes instead of purifying their own for every experiment. It would reduce variability and enable them to focus on the interesting biological questions rather than the requisite protein isolations needed to ask those questions.

Biotechnology and life science research have come a long way since then. We now take reliable, commercially available restriction enzymes for granted. Still, Promega keeps asking Bill’s original question: What can we do to make your work easier? The answers to that question have led to ground-breaking luciferase reporter technology, add-mix-measure assays for everything from cell viability to P450 activity to NADP/H detection, and standardized thaw-and-use primary cell lines for bioassays.

Today’s Difficult Reagent: Primary Antibodies

Among the most difficult reagents to work with and standardize are antibodies. If you are generating polyclonal primary antibodies that are not commercially available to study your target protein, you first need to find an animal with clean pre-immune serum. Usually that serum is evaluated by Western blot, which itself is an inherently fussy technique. And polyclonal antibodies are just that—polyclonal. What is generated after exposure to your antigen is a mixture of antibodies, and the composition of the mixture can change over time as the animal’s immune response matures. This gives you batch-to-batch variability and can make reproducibility more difficult. Commercially produced polyclonal antibodies are subject to the same exact issues. In August 2026, a news article in Nature reported that a metascientist had released a database of more than 18,000 questionable validation images across the catalogs of 15 antibody suppliers. All reflect the antibodies may not be performing like the scientists expect (Garisto 2026). Additionally, production of antibodies requires animal resources. High-quality, application validated antibodies are expensive as well. Using primary antibodies to study proteins is a significant pain point for the bench scientist.

It’s Not Just the Antibodies. It’s the Western Blots Too.

Tagged proteins or high-abundance exogenous expression of a target protein will not always reflect the endogenous protein behavior, localization or function. Large tags can interfere with folding and function; over expression of a protein can produce false protein interactions. Your results could be showing you artifacts resulting from a situation in your cells that is simply not biologically relevant.

If you are studying your target proteins (and other molecules) using Western blotting, post-translational modifications such as phosphorylation, ubiquitination, and cleavage shifts can complicate the interpretation of your results. Multiple bands on a blot can be hard to understand—is the protein cleaved? Is there cross-reactivity with your antibody to another protein? Is your sample degraded?

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From Chromosomes to the Brain: How HaloTag Is Expanding What We Can See

Posted on by Michele Arduengo, PhD
Artist's 3D concept of HaloTag with linker attaching to a target protein

Sometimes in the life sciences, it’s really tempting to look at a process like mitosis and think, “Wow, we really understand that,” and stop asking questions. In other cases, significant barriers like reliable expression of a non-endogenous reporter in an organ such as the brain is a challenge that seems impossible to overcome. However, those are precisely the moments when science and scientists need to push the boundaries of what we can see. In this post, I discuss two studies where researchers are literally seeing what could not be seen before by developing new methods for looking at their model systems.

In both papers discussed here, the HaloTag® protein and ligands were key to developing new methods allowing scientists to visualize biological phenomena that had been previously inaccessible. HaloTag® protein allows different functionalities to be linked onto a single genetic construct, rapidly and covalently under physiological conditions for experiments ranging from live-cell imaging to capture of protein interaction complexes (1). In the first study, HaloTag-CENP-A + JF635 ligand was the specific tool that let them directly see, in real time, that chromosome-attached microtubules pivot as the spindle elongates. In the second study, HaloTag was used to create a protocol for PET imaging of reporter gene systems in the brain.

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New Approach Methodologies: Guidelines for Biological Ground Truths

Posted on by Simon Moe

FDA Regulation changes: The Problem is still Validation

In March 2026, the FDA published draft guidance that fundamentally changed how NAMs (New Approach Methodologies) are evaluated in drug development. If you’re designing NAMs, whether that be spheroids, organoids, or organs-on-a-chip, your model now must meet specific validation criteria set by regulators. This draft document, titled ‘General Considerations for the Use of New Approach Methodologies in Drug Development’ provides requirements on the use of NAMs, including in vitro, in silico and in chemico methods (FDA & CDER, 2026). This guidance is a big shift from aspirational recommendations towards clearer regulatory recommendations.

It comes on the coattails of consistent feedback and challenges the market has seen over the past few years as they attempt to transition and optimize away from animal models. Mainly being:

“How can I be sure the data I get from this non-animal model is reliable, trustworthy and relevant?”

This regulatory mandate from the FDA requires researchers to use models and assays that meet four validation criteria:

  • Context of use
  • Human biological relevance
  • Technical characterization
  • Fit-for-purpose

This guidance initially applies to antibody development, biologics, and will eventually be relevant for small molecules. The momentum in the market is sound and indicates that there is a real need for assays that meet these new requirements.

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The Calcium-Platelet Connection in Cancer Metastasis 

Posted on by Anna Bennett

Most cancer-related deaths from solid tumors aren’t caused by the primary tumor itself, but are the result of metastasis. Metastasis is the process by which cancer cells break away from the primary tumor, travel through the bloodstream and establish new tumors in distant tissues. This is a complex, multi-step process and cancer researchers have spent decades trying to understand and disrupt the metastatic cascade. What is known: platelets and calcium play important roles in metastasis. What is unknown: How do these two factors interact?  

Here, we explore a recent study published in Scientific Reports by researchers at George Washington University1. They investigate how calcium levels influence platelet-cancer cell interactions and what happens when both factors converge.  

red blood cells,activated platelet and white blood cells microscopic photos
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Advancing Canine Cancer Immunotherapy through One Health Comparative Oncology Research

Posted on by Promega

The Case for Canine Immunotherapy

Cancer does not respect species boundaries. Each year more than four million dogs are diagnosed with cancer (1), making it the leading disease related cause of death in the canine population. Osteosarcoma, lymphoma, mast cell tumors and mammary carcinomas are among the most prevalent (1). In many cases, these tumors in dogs bear striking biological and molecular similarities to their human counterparts.

This convergence is the foundation of the Comparative Oncology (2) framework and One Health Initiatives. Companion pets, like dogs and cats, share our environments, our lifestyles and increasingly our therapeutic challenges. When research advances in veterinary oncology, it can open windows into human disease as well.

Comparative oncology integrates the advances and research of veterinary science. Dog and cat against a background of antibodies.
Comparative oncology integrates the advances and research of veterinary science, especially those of companion animals like dogs and cats, into more general oncology research, advancing the entire field of oncology.

What Veterinary Checkpoint Immunotherapy Brings to Comparative Oncology

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Charged Molecular Glues: The Molecular Trojan Horse That Slipped Past Biology’s Toughest Bouncer 

Posted on by Sara Millevolte

The cell membrane is notoriously selective about what it lets in. Charged molecules? Mostly rejected at the door. That’s a problem, because some of the most promising drug targets sit behind that barrier, and reaching them requires chemistry the membrane won’t tolerate. 

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Beyond the Liver: How Liposomal LNPs Are Expanding the Reach of mRNA Delivery

Posted on by Simon Moe

Introduction

Lipid nanoparticles (LNPs) have transformed mRNA delivery. From COVID-19 vaccines to the first approved RNAi therapeutic, ONPATTRO (Patisiran), LNPs have proven their ability to ferry nucleic acid cargo into cells with speed and efficiency (Huang, 2019). Despite this transformation, most clinically deployed LNP formulations share a significant constraint: following intravenous administration, roughly 90% of the injected dose clears to the liver within an hour. If your target is a hepatocyte, that is hardly a hindrance. It’s a serious limitation if you need to reach the spleen, lymph nodes, pancreas or other extrahepatic tissues, all of which are organs of major interest in immunotherapy, vaccine development and metabolic disease research.

A new paper from Pieter Cullis’s laboratory at the University of British Columbia (UBC) offers a structural solution to that problem. Their design, termed a ‘liposomal LNP’, reengineers the architecture of the particle itself to achieve dramatically longer circulation lifetimes and improved transfection in tissues that standard formulations have largely missed.

What are Lipid Nanoparticles?

LNPs enable fast transfection of a wide variety of cells, facilitating the transport of mRNA, DNA and siRNA into cells to induce transient expression in a short period of time (mere hours for mRNA and one to two days for DNA). They are a powerful tool that rose to broad public awareness through their use in COVID-19 vaccines, which delivered spike protein mRNA as cargo. Beyond vaccines, LNPs have been applied therapeutically as the delivery vehicle for ONPATTRO, which treats polyneuropathy of hereditary transthyretin-mediated amyloidosis (Huang, 2019).

The most widely studied LNP formulation, such as the ONPATTRO-like composition, consists of four components: an ionizable lipid, a helper lipid, cholesterol and a PEG-lipid. At physiological pH, the ionizable lipid is neutral and resides in a hydrophobic oil-like core surrounded by a lipid monolayer. This structure is highly effective at transfecting hepatocytes, but its rapid hepatic clearance limits its utility for reaching other tissues.

A Structural Redesign: What Makes the Liposomal LNP Different

Standard ONPATTRO-like formulations have a lipid monolayer surrounding an oil droplet core. The UBC team’s publication reasoned that dramatically increasing the proportion of bilayer-forming lipids, specifically equimolar egg sphingomyelin (ESM) and cholesterol, could fundamentally change what the LNP looks like (Cheng, 2025).

The authors explored various bilayer-to-ionizable lipid molar ratios (RB/I) to see how they modified the structure of the particle. They found that an RB/I of 4 resulted in particles that transition to a true liposomal architecture consisting of a lipid bilayer enclosing an aqueous interior with a solid core suspended inside. Cryo-electron microscopy confirmed that approximately 84% of particles at this ratio adopt this bilayer structure, with the solid core occupying roughly 30% of the interior. LNP sizes across all tested ratios remained in the 40–60 nm range, confirming that the structural shift happens without meaningful changes in particle size.

Why the Structural Change in Liposomal LNP Affects Assembly and Delivery

The liposomal LNP exploits pH-driven structural transitions at both the assembly and delivery stages, explaining how a particle dominated by bilayer lipids can remain transfection-competent.

Assembly: When an ethanol-lipid mixture meets an acidic aqueous buffer (pH 4) containing mRNA, the positively charged ionizable lipid binds the negatively charged mRNA, forming a core complex. This complex acts as a nucleation point for the deposition of ESM/cholesterol bilayer lipid. As pH rises to 7.4 during formulation, ionizable lipids in the outer monolayer shift to a neutral form and migrate inward, expanding an oil droplet core. The mRNA dissociates from the oil core and resides in the aqueous interior where it is protected within the bilayer.

Delivery: After endocytosis, the acidic endosomal environment reverses this process. The ionizable lipids become positively charged again and migrate to the outer surface of the LNP, causing the solid mRNA-containing core to extrude outward from the liposomal bilayer. This positively charged protrusion interacts with the negatively charged endosomal membrane, triggering fusion and releasing the mRNA into the cytoplasm for translation. The authors describe this as a localized “warhead” mechanism—a structural consequence of the bilayer architecture, rather than a simple membrane-disruption event.

Exploring the Performance of the Liposomal LNP

The authors utilized NanoLuc® mRNA as a reporter payload throughout the study. From in vitro transfection efficiency to whole-animal imaging, it allowed the authors to detect differential expression that would have been difficult to detect with less sensitive reporters.

The performance of the Liposomal LNP tells a compelling story. In vitro, the RB/I = 4 formulation matched or exceeded the transfection potency of the ONPATTRO-like composition in Huh7 cells across a wide dose range, while also proving to be the most stable on the shelf. After 63 weeks at 4°C, it maintained greater than 80% mRNA encapsulation with less than 20% size increase, and produced the highest mRNA integrity and translatability of any tested ratio.

SPECT/CT imaging of whole animals with the Liposomal LNP showed a circulation half-life approximately 15-fold longer than the standard ONPATTRO-like formulation, a direct consequence of the bilayer exterior adsorbing roughly half the plasma protein load. This reduced exterior plasma protein load means less macrophage recognition, less clearance and more time in circulation to reach tissues beyond the liver. That improved lifetime in circulation translated into improved tissue access. Ex vivo organ analysis showed 50-fold greater luminescence in the spleen and 150-fold greater in the inguinal lymph node compared to the standard formulation. Meaningful signal was also detected in the pancreas, a tissue rarely reached through conventional LNP formulations. Immunofluorescence confirmed delivery was localized to macrophages at the marginal zone of the spleen and subcapsular sinus of the lymph node.

It is also worth noting that the liposomal morphology held up when tested with the ionizable lipids used in the BNT162b2 and the mRNA-1273 COVID vaccines, suggesting this is a generalizable design.

Expanding Use of NanoLuc® mRNA: UBC RNA and Formulation Core

Throughout this study, NanoLuc® mRNA served as the reporter payload. In vitro, NanoLuc® luminescence normalized to total protein provided a sensitive, linear measure of transfection efficiency across a wide dose range. In vivo, it enabled whole-animal IVIS imaging using the Nano-Glo® Fluorofurimazine substrate, with quantification extended to ex vivo organ homogenates using the Nano-Glo® Luciferase Assay System. NanoLuc® Luciferase sensitivity enabled detecting differential expression in tissues as small as inguinal lymph nodes and the pancreas. Detecting meaningful signal from a lymph node or pancreas can be challenging and thus successful detection demonstrates the exceptional performance of NanoLuc® Luciferase.

The authors synthesized their NanoLuc® mRNA in-house, a capability not universally available to research groups. We have partnered with the University of British Columbia RNA and Formulation Core to close that gap, enabling distribution of NanoLuc® mRNA across the core’s academic and industry network. Researchers who want to investigate LNP delivery, optimize formulations or validate mRNA constructs can now work with the RNA and Formulation Core to acquire NanoLuc® mRNA without the overhead of in-house synthesis. Work from UBC has contributed foundational understanding for LNP formulations, and now through their core they enable NanoLuc® mRNA development for any interested scientist.

Conclusion

The work from UBC demonstrates what becomes possible when mRNA delivery can reach beyond the liver, but the findings are only useful if researchers can access the tools to replicate and build on them. That’s where the UBC RNA and Formulation Core comes in. By partnering with Promega to distribute NanoLuc® mRNA, the Core gives researchers direct access to the same reporter used in this study, without the overhead of in-house synthesis. Whether you’re optimizing an LNP formulation, validating extrahepatic delivery or exploring mRNA constructs for a new application, you can now work with the Core to get started.

Interested in learning more about the UBC RNA and Formulation Core? Explore their website.

Learn more about the full NanoLuc® portfolio.

Citations

Cheng, M.H.Y. et al. (2025) Liposomal lipid nanoparticles for extrahepatic delivery of mRNA. Nature Communications 16, 4135.
Huang, Y.Y. (2019) Approval of the first-ever RNAi therapeutics and its technological development history. Prog. Biochem. Biophys. 46, 313–322.

Stolen Chloroplasts and the Chrysalis of Complex Life 

Posted on by Elise Johnson

Despite its many mysteries, a chrysalis is one of the most familiar transformations in nature. We know what goes in. We know what comes out. For a long time, what happened in between was essentially invisible to us. Not because we weren’t curious, but because the mechanism was sealed inside something the size of a thumbnail, and we had no way in.

This same invisibility exists on a much older and much larger scale.

Sometime around two billion years ago, a cell swallowed a bacterium and, instead of digesting it, kept it alive inside itself. This process, called endosymbiosis, is arguably the single most consequential event in the history of complex life. The bacterium became a permanent resident, and over billions of years of co-evolution, it became something else entirely: the mitochondria that power every complex cell on earth. Without it, the living world as we know it doesn’t exist.

Scientists have known for decades that this kind of cellular acquisition had to have occurred. What has proved harder to explain is not that it happened, but how it started. What did the earliest molecular steps actually look like from the inside?

In the ocean, there is a microscopic single-celled organism called Rapaza viridis. It hunts algae by propelling itself through the water on whip-like appendages called flagella. That hunt may be showing us the beginning of a modern endosymbiosis: the same process that gave every complex cell its mitochondria and every plant its chloroplasts.

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New Study Suggests Cancer Research Has an Age Problem

Posted on by Kelly Grooms
White lab mouse among test tubes overlaid with a blue DNA helix.

Most cancer research relies on young, healthy mice. Most cancer patients are not young. Could this disconnect between model organism and patient, which ignores the impact of the physiological realities of aging, explain why some therapies perform well in the lab but not in clinical trials? Focusing on lung cancer, a study published in Nature investigated what happens when these two realities, young and old, finally meet in the lab (1). The answers could reshape how we think about cancer research and the impact of aging on metastasis and disease progression.

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Before the First Dose

Posted on by Elise Johnson

Kierkegaard observed that one of humanity’s enduring tensions is that while life can only be understood backwards, it must be lived forwards. It’s a truth medicine knows intimately: in the treatment that worked until it didn’t, the resistance that arrived without warning, the moment a doctor has to tell a patient that the drug that was helping has stopped. Not because anyone made a mistake, but because the critical knowledge that would have mattered arrived too late, if at all.

A recent paper from the National Cancer Institute is, in a small but meaningful way, science’s pursuit of that elusive foresight: an understanding that emerges early enough, for once, to change what happens next.

The Elegant Idea

For decades, chemotherapy has worked by brute force, flooding the body with toxins designed to kill rapidly dividing cells. The problem is that rapid division isn’t unique to cancer. Hair follicle cells, gut lining cells and immune cells also divide rapidly, which is why patients lose hair, lose energy and become susceptible to infection. Chemotherapy targets a behavior, but the drug has no way to tell a healthy cell from a cancerous one.

Antibody-drug conjugates (ADCs) change that. Instead of targeting what cancer cells do, they target what cancer cells are. Cancer cells tend to display certain proteins on their surface in far greater numbers than healthy cells do. The antibody is engineered to seek out those proteins specifically. It navigates to its target, binds and waits for the cell to do what cells routinely do: pull it inside. Once there, the cell’s own digestive machinery (the lysosome) breaks down the chemical tether holding the toxin to the antibody, releasing the toxin to kill the cell from within. More than a dozen ADCs have received FDA approval in recent years, and the field is evolving fast.

What the Cell Does Next

But cancer cells don’t simply accept their fate. Even when an ADC delivers its payload perfectly—the antibody finds its target, the cell pulls it inside, the lysosome cuts the tether—a pump embedded in the cell membrane can grab the released toxin and throw it back out before it causes damage.

The delivery worked. The package got ejected anyway.

These pumps—ATP-binding cassette transporters, or more plainly, efflux pumps—are a normal feature of cell biology. Their job is cellular housekeeping, clearing out unwanted or toxic substances before they cause damage. Under the pressure of drug treatment, cancer cells do what life has always done under pressure: the ones best equipped to survive do. The same mechanism that has shaped living things for billions of years now works against the treatment. Not all cancer cells are identical, and the ones that happen to produce more pumps survive while others don’t, gradually shifting the tumor toward resistance.

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