Protein Analysis and Purification

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?

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

ADC Development: The Questions Running in the Background

Posted on by Elise Johnson

You’ve had food stuck in your teeth at some point during a conversation you thought was going well. Or toilet paper trailing from the back of your shoe on a day you felt particularly put together. Maybe you’ve even forgotten to color in your very blonde eyebrows and spent the rest of the day looking like someone erased the top half of your face. But you had no idea. You’re walking around with the quiet, complete confidence of someone operating on incomplete information.

You weren’t wrong about anything you could see. You just couldn’t see everything, which is a different problem than getting a bad result. A bad result tells you something is wrong, but this doesn’t. This happens in the lab too. The number is clean, the program is advancing, and somewhere in the data, something is happening that your readout has no way to show you.

Antibody-drug conjugate (ADC) development has a version of this problem. The cytotoxicity readout is real, reliable, and correct. It’s also an aggregate, and an aggregate compresses everything that happened into a single number. That number can’t tell you which mechanisms produced it, which ones are underperforming, or what to change if the program stops working. It just tells you cells died, or they didn’t. You’re walking around with the quiet, complete confidence of someone operating on incomplete information.

Cytotoxicity: Where Every Program Begins

Cell viability assays were built to answer one question: did the cells die? Increase the dose, more cells die. Decrease it, fewer do. The curve is clean, the data is reliable, and the payload is doing what it was designed to do. For cytotoxicity, it’s the right question to ask.

That question has an established platform with consistent, reproducible data: our CellTiter-Glo® and RealTime-Glo™ Assays.

For most ADC programs, this is where the measurement work starts and stops, but it should only be where it starts. The question they answer well is only one of several your ADC is raising. The number means what it says, but the question it answers has a boundary, and the boundary leaves you with an incomplete picture.

Continue reading “ADC Development: The Questions Running in the Background”

Another Crack in the RAS Code: The Measurement Story Behind Daraxonrasib

Posted on by Elise Johnson

In late May 2026, a clinical trial result landed in the New England Journal of Medicine and immediately rewrote what oncologists believed was possible for patients with metastatic pancreatic cancer. Before the paper was published, people in the field were already calling it “transformative.” The data, when it came, agreed. In a disease where most second-line treatments offer months at best, a drug called daraxonrasib nearly doubled how long patients lived compared to those who received chemotherapy.1 It was approved by the FDA in August 2026 as RASONQUE, six months ahead of schedule.2

RAS proteins, which regulate cell growth and are mutated in more than a third of all human cancers,3 had spent forty years resisting every attempt to drug them. The protein’s surface offered no obvious foothold for a small molecule. Once the word “undruggable” attached itself to the RAS protein family, most of the field moved on to more cooperative targets.

Some researchers stayed. And Promega stayed committed to the question that never goes away: does this new compound work inside a living cell? When the next chapter of the RAS story arrived, the tools were ready. Daraxonrasib is one culmination of a much longer story, one that matters for every researcher pursuing a target the field has written off.

The First Answer

To understand what daraxonrasib represents, it helps to see how an earlier chapter of the RAS story faced the same fundamental measurement challenge.

Continue reading “Another Crack in the RAS Code: The Measurement Story Behind Daraxonrasib”

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.

Continue reading “CRISPR/Cas9 Knock-In Tagging: Simplifying the Study of Endogenous Biology”

A New View of Protein Degradation with HiBiT and Live Cell Imaging

Posted on by Anna Bennett

Targeted protein degradation (TPD) is an emerging drug discovery strategy that offers an entirely different approach to tackle disease-relevant proteins, including classic “undruggable” targets. Instead of inhibiting protein function, small molecules like PROTACs and molecular glues co-opt the cell’s own ubiquitin-proteasome system to eliminate specific proteins altogether. But as this targeted approach gains traction, it also challenges existing methods for validating compound activity.  

How do you confirm that degradation is happening in a biologically-relevant system? Can you validate protein degradation in real-time?  

Continue reading “A New View of Protein Degradation with HiBiT and Live Cell Imaging”

Understanding Wnt Signaling Through β-Catenin Localization in Live Cells

Posted on by Anna Bennett

The Wnt/β-catenin pathway, long studied in the context of developmental biology, has become increasingly recognized for its role in a wide range of human diseases. Its dysregulation has been implicated in cancer, fibrosis, immune modulation, and neurodegenerative conditions—making it a clinically actionable target across diverse therapeutic areas1. In this blog, we cover the fundamentals of Wnt/β-catenin signaling, highlight ongoing research efforts to understand its role in disease, and show how combining live-cell imaging with luminescent assays complements functional studies.

Continue reading “Understanding Wnt Signaling Through β-Catenin Localization in Live Cells”

Do Zebrafish Hold the Key to Heart Regeneration? 

Posted on by Shannon Sindermann
Heart regeneration after heart attacks can stop poor health outcomes, read for more research

The human body has an incredible capacity for self-repair. Our skin can regenerate after a small cut, bones can heal after fractures and even the liver can regrow to its original size after 70% is lost or removed (3). However, when it comes to the heart, the story is very different. As Miley Cyrus once sang, “nothing breaks like a heart” – and science agrees. Unlike other organs, the heart has almost no ability to regenerate itself after injuries. In instances like myocardial infarctions, more commonly known as heart attacks, large amounts of cardiomyocytes (CMs)—the cells responsible for heart muscle contraction—are lost and cannot be regenerated, causing the formation of non-regenerative fibrotic scar tissue and, ultimately, decline in heart function (1).  

Continue reading “Do Zebrafish Hold the Key to Heart Regeneration? “

Unlocking the Secrets of ADP-Ribosylation with Arg-C Ultra Protease, a Key Enzyme for Studying Ester-Linked Protein Modifications 

Posted on by Michele Arduengo, PhD

Post-translational modifications of proteins are critical for proper protein function. Modifications such as phosphorylation/dephosphorylation can act as switches that activate or inactivate proteins in signaling cascades. The addition of specific sugars to membrane proteins on cells are critical for recognition, interaction with the extracellular matrix and other activities. While we know volumes about some types of protein modifications, ADP-ribosylation on aspartate and glutamate residues has been more difficult to study because of the chemical instability of these ester-linked modifications. 

Matić Lab (Eduardo José Longarini and Ivan Matić) recently published a study that explored mono-ADP-ribosylation (ADPr) on aspartate and glutamate residues by the protein PARP1 and its potential reversal by PARG. PARP1 and PARG signaling are central to DNA repair and apoptosis pathways, making them potentially powerful therapeutic targets in cancer or neurodegenerative diseases in which DNA repair processes are often disrupted. 

Continue reading “Unlocking the Secrets of ADP-Ribosylation with Arg-C Ultra Protease, a Key Enzyme for Studying Ester-Linked Protein Modifications “

Fluorescent Ligands in Biological Research: Where We’ve Been, Where We’re Headed

Posted on by Kari Kenefick

Fluorescent tags (fluorophores), have become excellent tools for labeling cells and cellular components. They can be used for imaging large molecules like proteins, on down to cellular components and enzymes such as transcription factors. Once labeled, these molecules can be tracked in tissue or inside a cell, when the right tag is used.

What is the ‘right’ tag? It’s a tag with bright signal, with low background and good photostability. For small cell components like organelles, the tag must be cell-permeable and small enough to not interfere with normal cellular processes such as transcription and metabolism.

Significant advances have been made in fluorescent tags in the past two decades. Here we look at several papers noting these advances.

Continue reading “Fluorescent Ligands in Biological Research: Where We’ve Been, Where We’re Headed”

Designing Better Therapeutic mAbs: An Assay for Rapid, Parallel Screening of Fc/ FcɣR Interactions

Posted on by Michele Arduengo, PhD

The first monoclonal antibody (mAb) was produced in a lab 1975, and the first therapeutic mAb was introduced in the United States to prevent kidney transplant rejection in 1986. The first mAb used in cancer treatment the anti-CD20 mAb, rituximab, was used to treat non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. Today therapeutic mAbs have become a mainstay of cancer, autoimmune disease, and metabolic disease therapies and include HERCEPTIN® used to treat certain forms of breast cancer, Prolia used to treat bone loss in post-menopausal women, and Stelara used to treat autoimmune diseases like psoriatic arthritis and severe Crohn disease, among many others. Therapeutic mAbs bind targets with high specificity and affinity and they can recruit effector cells to drive target elimination through mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), making them highly specific, effective therapies.

3D rendering of a Lumit Assay which can be used for plate-based screening assay to measure the affinities of Fc interactions of therapeutic mAbs.
Continue reading “Designing Better Therapeutic mAbs: An Assay for Rapid, Parallel Screening of Fc/ FcɣR Interactions”