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Detect Phosphorylation of Proteins with RePlex

Posted October 21, 2021

"By switching my assays over to Simple Western, I am able to increase my throughput, allowing for full dose responses to be generated in each experiment. Utilizing the RePlex module even further increases this throughput as I often probe for 5-6 proteins per capillary in a single run including phosphorylated/total protein pairs in the same lane."

- Joseph Klebba, Ph.D., Scientist, Vividion Therapeutics

Transforming Human Health One Small Molecule at a Time

Vividion Therapeutics is a small molecule company harnessing its expertise in target biology, chemistry, and mass spectrometry to transform the future of human health. They target disease-critical proteins that were once considered inaccessible with unmatched precision that limits the risk of off-target side effects. Dr. Joseph Klebba’s interests are in the oncology/immunology therapeutic areas where he drives pre-clinical studies focused on inhibiting a key signaling pathway implicated in tumorigenesis.

Measure Compound Efficacy Using In Vitro and In Vivo Models

To assess the ability of the small molecule to inhibit the pathway of interest, Joe runs dose response curves to look for changes in the phosphorylation state and the abundance of key proteins using in vitro and in vivo models. However, monitoring quantitative phospho-protein changes is notoriously problematic and slow. High-throughput techniques like ELISA and HTRF are often unreliable and traditional Western blots, the best method for detecting phospho-proteins, are time-consuming, low-throughput, and often don’t have the sensitivity required to detect low levels of phosphorylated proteins.

By transferring his assays over to Simple Western™ with Jess™, Joe now can measure compound efficacy, in both in vivo and in vitro models, in a way simply not possible with standard methods. Generating a full dose-response curve in a single 3 hour experiment is now possible, compared to the 2-3 days it would have taken using traditional Western blot. Jess’ throughput is far superior to a traditional Western blot, allowing Joe to generate comprehensive, reliable data in a shorter amount of time to accelerate compound progression and move the project forward faster.

Joe can push his throughput even further by using Jess’ RePlex™ module, which allows him to analyze as many as five to six proteins in a single capillary without having to worry about the signal loss that is observed when stripping and re-probing a traditional Western blot. This means he can measure the levels of phosphorylated target, the overall abundance of the target, total protein for sample normalization, and downstream proteins or viability indicators in the same sample.

Accurately Quantify the Phosphorylation of Proteins with RePlex

Not only does the RePlex module increase Joe’s throughput, it also improves the accuracy of his phospho-protein quantification. Normalizing against a standard loading control, which can be variable between samples, adds noise when it comes to determining what percentage of the protein of interest is phosphorylated. With the RePlex module, Joe can first probe for phosphorylated protein levels and then, in the same capillary, probe for total target levels to accurately calculate the percent phosphorylation. He can also kick up his accuracy by normalizing his data against the total protein in the sample to normalize out any variability in sample concentration during loading.

Jess is also more sensitive compared to traditional Western blot, which also adds to the reliability of the data generated. The signal strength of phosphorylated proteins can be low and is often close to the limit of quantitation (LOD) for traditional Western blots. This can negatively impact the reliability and reproducibility of the data generated. Jess is more sensitive than traditional Western blots which translates to more reliable and robust quantitation of low abundance targets. This has been particularly enabling when measuring compound efficacy using tissues collected from in vivo studies.

RePlex Frees Up Your Time in the Lab

Besides the throughput and quantitation advantages Joe has gained with Jess, Simple Western has also freed up his time in the lab. He is able to set up a run and then let Jess take over to deliver fully analyzed results in 3 hours. When asked what he does with all his extra free time, Joe replied “I have two young children; I am no longer familiar with the concept of free time.” Nevertheless, Simple Western’s hands-free automation and rapid time to results have given him the flexibility to spend his time working on other experiments or spending time with his family.

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