Mary Makes More Links Between Obesity and Cancer with NanoPro 1000
"The Simple Western Charge assay, a capillary isoelectric focusing immunoassay, exceeded the reliability of 2D Western blots for resolving recombinant PKG-Iα and PKG-Iβ and uses 100,000X less sample quantity, making it well-suited for clinical disease proteomics because protein isoforms and post-translational modifications can be detected in precious tissue biopsies.""
- Mary G. Johlfs, M.S., Director of Research Operations/Scientist, Roseman University of Health Sciences
PROFILING THE PROTEOME
Mary G. Johlfs is the Director of Research Operations and a scientist at Roseman University of Health Sciences. She oversees the day-to-day functionality of expanding research groups at Roseman, collaborating with Roseman principal investigators as well as with scientists from other prominent institutes and universities. She contributes to profiling the cellular proteome to better understand disease pathogenesis of diabetes, obesity, neurodegenerative diseases as well as mesothelioma, lung and ovarian cancers.
Research efforts have been expanded to understanding the link between cancer and diabetes/obesity. For example, three different protein kinases have been simultaneously profiled, including cGMP-dependent protein kinase (PKG-I), a critical protein involved in understanding the role of nitric oxide (NO) in fat (adipocyte) differentiation. A better understanding of fat cell differentiation will give researchers clues on clinical ways to address obesity, a condition recognized in 1997 by the WHO as a global epidemic.
LIMITS TO 1D AND 2D WESTERNS
PKG-Iα and its alternate splice variant PKG-Iβ are activated by different levels of nitric oxide (NO). Low levels of NO activate PKG-Iα, promoting cell survival and proliferation, while high levels activate PKG-Iβ and inhibit cell proliferation. 1D Western blots didn’t give Mary and her colleagues the resolution needed to separate PKG-Iα (76.4 kDa) from PKG-Iβ (77.8 kDa). There also weren’t many isoformspecific pan and phospho PKG antibodies to choose from, and the few antibodies they could get often cross-reacted with other proteins.
2D Westerns, which separated proteins first by isoelectric point and then by size, gave them the separation and post-translational modification (PTM) information they needed but required 1 µg of recombinant protein for analysis, limiting the amount of data they could get from tissue biopsies. The 2D Westerns were also technically challenging to run — to the point where Mary and her colleagues were sending their samples out to run at a third-party lab.
GETTING THE MOST OUT OF EVERY SAMPLE
When Mary and her colleagues tried Simple Western™ Charge assays on the NanoPro™ 1000, they were able to separate PKG-Iα and PKGIβ solely based on their isoelectric point and finally got the separation they needed to distinguish between the two isoforms. They could also monitor PTMs without a phosphospecific antibody as phosphorylation events change the isoelectric point of a protein and cause them to migrate differently from nonphosphorylated protein.
More importantly, the Simple Western Charge assay was 100,000X more sensitive compared to a 2D Western! Only 10 pg of recombinant PKG-I was needed to get results compared to 1 µg for the 2D Western (Figure 1). With this new ability to resolve PKG‑Iα and PKG-Iβ with very small sample requirement, Mary and her colleagues compared expression of each isoform in various tissue types and found that they respond differently to the biological effects of NO. They were also the first to report different expression levels of the two isoforms at different stages of adipocyte differentiation, something they wouldn’t have been able to discover with other technologies where the isoforms co-migrate.
FIGURE 1. Recombinant PKG-Iβ and PKG-Iα run on 2D Western (left) and the Simple Western Charge assay on the NanoPro 1000 (right). Both techniques resolved the two isoforms but NanoPro 1000 required 100,000X less sample compared to the 2D Western.
NEXT STEP: BIOMARKERS FOR DEMENTIA
Simple Western Charge assays on the NanoPro 1000 changed how Mary and her colleagues profile the proteome since they can now resolve isoforms like PKG-Iα and PKG-Iβ and monitor proteins using extremely small quantities of precious sample. She now plans to use Simple Western Charge assays to define biomarkers that will distinguish among different dementia-related diseases to aid in more accurate diagnosis and treatment.
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MG Johlfs et al. (2015) Capillary isoelectric focusing immunoassay for fat cell differentiation proteomics PLoS One e0132105.. PMID: 26132171.
RR Fiscus et al. (2012) Protein kinase G (PKG): Involvement in promoting neural cell survival, proliferation, synaptogenesis, and synaptic plasticity and the use of new ultrasensitive capillary-electrophoresis-based methodologies for measuring PKG expression and molecular action Protein Kinase Technologies, NEUROMETHODS series (Springer) 319.