For well over four decades, the laboratory technique called the western blot has been an important part of research in all fields related to proteomics (the large-scale study of proteins). But researchers continue to find innovative applications for this stalwart protein detection method.
Proceeded by southern blotting (which aids in DNA mapping by detecting any gene of interest in a given sample), western blotting employs similar methods to detect specific protein molecules and differentiate them from the mixture of various proteins that surround them. Blotting techniques such as western and southern blotting get their name from a key procedural step that involves transferring prepared proteins from a special gel to a blotting membrane.
In the case of the western blot, the blotting stage is preceded by a chemical protein separation process and a gel electrophoresis process that sorts each protein by size. After their transfer to the blotting membrane, they undergo a series of treatments to identify specific proteins of all tissue and cell types.
Because it has become such a commonplace research tool, the western blot is easy to overlook when it comes to driving innovation. However, clinical advancements in the fields of fluorescence and chemiluminescent reagents have expanded the capabilities of the western blot, lending it new relevance in some of the most essential and fastest-growing fields of modern medical research.
In the field of pain management, for example, the western blot played an invaluable part in a recent study of neuropathic pain caused by central or peripheral nerve damage. Authored by researchers from Nanjing Medical University in Jiangsu, China, this study appeared in the British Journal of Anaesthesia.
To reach its objective of determining what role the tyrosine kinase-like receptor ROR2 (orphan receptor 2) might play in neuropathic pain regulation, the Nanjing team drew upon the relatively simple and reliable ability of the western blots to detect protein modifications. Working with mice, the study ultimately discovered that ROR2 mRNA was increased in spinal neurons in the wake of chronic constriction injury (CCI). Furthermore, the tyrosine phosphorylation of ROR2 experienced a dramatic boost over the days that proceeded the CCI.
The protracted study further demonstrated that the elimination of ROR2 in the spinal cord using siRNA effectively reversed observable CCI-induced pain behaviors. In contrast, the upregulation of spinal ROR2 in mice induced pain behaviors. In short, western blot analysis drove a significant step forward in the study of ROR2-mediated pain modulation.
Western blots are also playing a vital role in trailblazing cancer research. Last year, the researchers at Germany’s University of Cologne published a study in Technology in Cancer Research and Treatment that established a promising application for western blot analysis when it comes to identifying mutation of the IDH1 gene in relation to the development of progressive gliomas.
Researchers performed DNA sequencing on 113 tumor samples from 84 glioma patients and then assessed the qualitative expression of IDH1 in those samples using western blot analysis. They also used real-time polymerase chain reaction (real-time PCR) to assess IDH1 mutation expression quantitatively.
Researchers ultimately discovered that western blot analysis can detect IDH1 mutations with perfect or near-perfect sensitivity and specificity. Western blot analysis bested real-time PCR assessment by 6% in terms of sensitivity and 2% in terms of specificity.
Beyond cancers of the nervous system, western blotting holds tremendous therapeutic promise for any number of cancer types by helping researchers pinpoint the genetic factors that underly tumorigenesis in general. In fact, the potential applications of the western blot extend far past few the oncological and pain management examples provided here.