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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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Screening Disease-Relevant Biology: How the Endo-GeneScreen Platform Uses Endogenous Protein Detection to Drive Drug Discovery 

Posted on by Amy Landreman

Drug discovery has long grappled with a fundamental tension in high-throughput screening: the more biologically relevant your model, the harder it is to scale. Phenotypic assays in primary disease-relevant cells offer rich biological context, but capturing meaningful, target-specific readouts from these complex systems at screening scale has remained a significant challenge.  In contrast, simpler, more scalable systems are easier to deploy but sacrifice the biological fidelity that makes hits meaningful. A recent study by Samowitz et al. in Nature Communications describes an interesting approach to resolve this tension, the Endo-GeneScreen (EGS) platform. A high-throughput screening system designed to enable scalable detection of endogenous protein levels within disease-modeling cellular contexts. 

A Well-Chosen Proof of Concept 

The authors selected Syngap1 as their proof-of-concept target to develop and demonstrate this approach. De novo mutations in this gene that lead to haploinsufficiency are among the most common genetic causes of sporadic neurodevelopmental disorders, including intellectual disability, autism, and epilepsy. Small molecules that boost SynGAP protein levels back toward wildtype would address the root cause of these disorders rather than managing downstream symptoms. Importantly, Syngap1 function is closely tied to cortical excitatory neurons. Well-validated in vitro and in vivo models for these neurons already exist, creating an integrated system for both discovering new compounds and validating them in the same biological context. That continuity is an important step toward improving the translational relevance of lead molecules coming out of the screen. 

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Promega Fc Effector Assays: Measure Every Mechanism

Posted on by Promega

This post is written by Kai Hillman, PhD, Promega Corporation.

Every day, scientists push the boundaries of what’s possible with monoclonal antibodies (mAbs)—from targeting cancer cells to calming autoimmune-driven inflammation. These therapies rely not only on binding but on engineering the desired immune response. The suite of Promega Fc Effector Assays helps you understand these interactions from receptor binding and function, through bridging studies. With consistency, sensitivity, and scalability, these assays support teams from early discovery through lot release.

This article draws on real-world publications and product insights to show how Promega assays are powering next-generation immunotherapies—and redefining how we measure immune engagement.

Schematic diagramming the suite of Promega Fc effector assays in one seamless workflow to support antibody development across the pipeline.
Figure 1. Promega delivers the most comprehensive suite of Fc effector assays in one seamless workflow to support antibody development across the pipeline.
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CRISPR/Cas9 Knock-In Tagging: Simplifying the Study of Endogenous Biology

Posted on by Ken Doyle

Understanding the expression, function and dynamics of proteins in their native environment is a fundamental goal that’s common to diverse aspects of molecular and cell biology. To study a protein, it must first be labeled—either directly or indirectly—with a “tag” that allows specific and sensitive detection.

Using a labeled antibody to the protein of interest is a common method to study native proteins. However, antibody-based assays, such as ELISAs and Western blots, are not suitable for use in live cells. These techniques are also limited by throughput and sensitivity. Further, suitable antibodies may not be available for the target protein of interest.

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Virus-Like Particles: All the Bark, None of the Bite

Posted on by Ken Doyle

Globally, there have been over 5 million deaths attributed to COVID-19 since the start of the pandemic. Throughout the ongoing battle against SARS-CoV-2, researchers have been studying the viral lineage and the variants that are emerging as the virus evolves over time. The more opportunities that the virus has to replicate (i.e., the more people it infects), the greater the likelihood that a new variant will emerge.

This short video from the World Health Organization explains how viral variants develop.

The US Centers for Disease Control and Prevention (CDC) classify SARS-CoV-2 variants into four groups: Variants Being Monitored (VBM), Variants of Interest (VOI), Variants of Concern (VOC) and Variants of High Consequence (VOHC). So far, no variants in the US have been identified as VOHC or VOI. Currently, the most common variant in the US is the Delta variant (which includes the B.1.617.2 and AY viral lineages), and it is classified as a VOC.

The Delta variant originated in India and spread rapidly across the UK before making its way into the US (1). Current vaccines, including mRNA and adenoviral vector vaccines, have demonstrated effectiveness against the Delta variant. However, it is a VOC because it is more than twice as contagious as previous variants, and some studies have shown that it is associated with more severe symptoms.

A recent study (2) provides one explanation for the higher infectivity of the Delta variant, using an approach based on virus-like particles (VLPs). The research team was led by Dr. Jennifer Doudna, 2020 Nobel Prize winner for her work on CRISPR-Cas9 gene editing, and Dr. Melanie Ott, director of the Gladstone Institute of Virology at the University of California–Berkeley.

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Bringing Cutting Edge Technologies to Academic Researchers Through the Academic Access Program

Posted on by Promega

This post was written by guest blogger Iain Ronald, Director Academic/Government Market Segment at Promega.

My back story is similar to most of you reading this blog, high school education, undergraduate degree then onto a postgraduate degree. However, over 25 years ago during my undergraduate study, I was fortunate enough to work in the lab of Professor Ray Waters studying DNA damage in S. cerevisiae as a model organism and at the time PCR was cutting-edge technology and the PCR license was in full effect. However, there was one company that was fighting the good fight to democratize PCR for the good of the scientific community, Promega.

I became enamored with Promega then, and the next steps in my career were taken with a view to working at this company who, for all intents and purposes, seemed to really care about the progression of science beyond self-aggrandizement.

Now that I am working at Promega in a position where I can bring benefit to our academic community, I have pondered what I can do to equal the disruptive attitude I observed in this company all those years ago when they were fighting the then “big tech” for the enablement of the scientific community. 

Reporter bioassays are one of hte many offerings of the academic access program.
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A Closer Look at C. difficile Biology with Luminescent Tagging

Posted on by Jordan Villanueva

Clostridium difficile is a bacterium that infects around half a million people per year in the United States. The infection causes symptoms ranging from diarrhea to severe colitis, and it’s one of the most common infections contracted while staying in the hospital. As the global incidence of C. diff infection has risen over the past decade, so has the pressure to develop novel therapeutic strategies. This requires a thorough exploration of all aspects of C. difficile biology.

Two recent papers published by researchers at the University of Leiden have shed light on C. difficile physiology using HiBiT protein tagging. The HiBiT system allows detection of proteins in live cells using an 11 amino acid tag. The HiBiT tag binds to the complementary LgBiT polypeptide to reconstitute the luminescent NanoBiT® enzyme. The resulting luminescence is proportional to the amount of HiBiT-tagged protein that is present.

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NanoLuc® Luciferase: Brighter Days Ahead for In Vivo Imaging

Posted on by Ken Doyle
nanoluc in vivo imaging

NanoLuc® luciferase gave researchers a new way to see endogenous biology: how proteins behave in their native environments within cells and tissues. This small (~19kDa) enzyme, engineered from a luciferase found in the deep-sea shrimp Oplophorus gracilirostris, is up to 150X brighter than firefly or Renilla luciferase. For an overview of NanoLuc® luciferase applications, see: NanoLuc® Luciferase Powers More than Reporter Assays.

The small size of NanoLuc® luciferase, as well as the lack of a requirement for ATP to generate a bioluminescent signal, make it particularly attractive as a reporter for in vivo bioluminescent imaging, both in cells and live animals. Expression of a small reporter molecule as a fusion protein is less likely to interfere with the biological function of the target protein. NanoLuc® Binary Technology (NanoBiT®) takes this approach a step further by creating a complementation reporter system where one subunit is just 11 amino acids in length. This video explains how the high-affinity version of NanoBiT® complementation (HiBiT) works:

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CRISPR/Cas9 HiBiT Knock-In: A Scalable Approach for Studying Endogenous Protein Dynamics

Posted on by Darcia Schweitzer

Studying protein function in live cells is limited by the tools available to analyze the expression and interactions of those proteins. Although mass spectrometry and antibody-based protein detection are valuable technologies for protein analysis, both methods have drawbacks that limit the range of targets and contexts in which proteins can be investigated.

Mass spectrometry is often poor at detecting low-abundance proteins. Antibody-based techniques require high quality, specific antibodies, which can be difficult to impossible to acquire. Both methods require cell lysis, preventing real-time analysis and limiting the physiological relevance, and both methods can be limiting for higher-throughput analysis. While plasmid-based overexpression of tagged target proteins simplifies detection and can allow for real time analysis, protein levels don’t typically resemble endogenous levels. Overexpression also has the potential to create experimental artifacts or limit the dynamic range of an observed response.

In 2018, Promega R&D scientists published a paper in ACS Chemical Biology demonstrating the use of CRISPR/Cas9 to integrate the 11 amino acid, bioluminescent HiBiT tag directly into the genome to serve as an easily measured reporter for endogenous proteins. This provides a highly quantitative method for investigating cellular protein dynamics that sidesteps the need for cloning and other drawbacks to conventional methods, including the ability to measure changing protein dynamics in real-time. (For more details about CRISPR/Cas9 knock-in tagging and other applications, read this blog.)

While their findings showed that this method provides efficient and specific tagging of endogenous proteins, the research was limited to just five different proteins within a single signaling pathway in two cell lines. This left unanswered questions about whether this approach was scalable, had broader applications and how accurately the natural biology of the cells was represented.

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NanoLuc® Luciferase Powers More than Reporter Assays

Posted on by Sara Klink
Bright NanoLuc® Luciferase

What happens when you engineer a small, super bright luciferase? A generation of new tools. We’ve highlighted many of the papers and new applications that NanoLuc® luciferase has enabled on this blog. Introduced first as a reporter enzyme to assess promoter activity, NanoLuc® has since become the foundation for bioluminescent tools that reveal endogenous protein dynamics, target engagement, protein degradation, immunodetection and more. Much of that biology was largely invisible before these tools existed. So where did NanoLuc® come from, and how does one enzyme power so many research capabilities? Read on for a primer on the technologies and applications built from this enzyme over the last decade.

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