Distinguish PKG Antibody Isoforms with Simple Western

Finding More Links Between Obesity and Cancer with NanoPro 1000

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 understand better disease pathogenesis of diabetes, obesity, neurodegenerative diseases as well as mesothelioma, lung, and ovarian cancers.

Research efforts have been expanded to understand 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 weren’t many isoform-specific pan and phospho-specific PKG protein antibodies to choose from, and any PKG antibody 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 PKG 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 using a single PKG antibody. They could also monitor PTMs without a phospho-specific antibody as phosphorylation events change the isoelectric point of a protein and cause them to migrate differently from non-phosphorylated protein.

More importantly, the Simple Western Charge assay was 100,000X more sensitive than a 2D Western! Only 10 pg of recombinant PKG protein 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 by isoelectric point with very small sample requirement, Mary and her colleagues compared the 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 PKG protein isoforms at different stages of adipocyte differentiation, something they wouldn’t have been able to discover with other technologies where the isoforms co-migrate.