A novel high-throughput protein analysis approach utilizing a bead-based microarray platform
The Western blot has been a staple in the molecular biology tool kit for nearly 40 years. While at Stanford, George Stark was the first to describe the transfer of proteins by capillary action from a gel onto diazobenzyloxymethyl-paper, submitting his work to the Proceedings of the National Academy of Sciences (PNAS) in April 1979.
Simultaneously, Harry Towbin was working to determine the specificity of antibodies against proteins in complex macromolecular structures and unknowingly developed the same gel electrophoresis method, but with a transfer onto nitrocellulose. Towbin submitted his version of the method to PNAS in June 1979.
Only when both articles were published in the September issue of PNAS did the authors realize they had developed the same method.
But it was a third man, W. Neal Burnette, who was the first to coin the term Western blot – in homage to the Southern and Northern blots – noted at the end of the introduction section in his 1981 publication in the journal of Analytical Biochemistry.
Flash forward to today and Western blot analysis is practiced in the same analog format and remains as laborious and resource-intensive as it was back in 1979.
Markus Templin and colleagues from the NMI Natural and Medical Sciences Institute at the University of Tübingen, have developed the next generation of the Western blot. Known as DigiWest®, it enables a highly parallel analysis of protein expression and phospho-status by adapting the classical Western blot to a bead-based microarray platform. This novel protein profiling immunoassay is described in Nature Communications and is also available in a fee-for-service model through NMI TT Pharmaservices.
Inspired by the unparalleled size resolution of the classical Western blot, but frustrated by the lack of throughput, Markus Templin and his team set out to incorporate protein size information into their multiplex immunoassays. With DigiWest they’ve achieved an attractive alternative to currently available approaches, such as mass spectrometry, reverse-phase protein microarrays (RPPA), and next-generation Western blots based on:
- Enhanced spatial resolution
- Reduced sample requirement (as little as 50 μg) and quantitative phospho-status
- Enhanced sensitivity – Able to detect 10 pg/μg with a 1,000 fold dynamic range
- Increased throughput – Run 60 to 600 analytes per sample with 2 to 60 samples per study
- Automated workflows and digital data output
Challenges are inherent to developing a new method, but Templin and his team weren’t deterred with DigiWest. To develop and verify it against Western blotting, they established a panel of over 1,000 commercial antibodies.
Sounds too good to be true? Templin’s group validated the novel method with a series of four experiments:
- DigiWest exhibited comparable sensitivity and linearity during a spike-in experiment but required only 1/100th of the initial sample as compared to a traditional Western blot. Additionally, the intra-assay variation (CV of replicate measurements from the same bead-set) was below 5%.
- Highly similar expression patterns were observed as compared to a mass spectrometry approach that used Kinobeads to identify a set of 24 kinases, which showed significant differences between Lapatinib-resistant and the H292 parental cell line.
- Major changes in the expression levels of Ephrin-receptors were revealed, indicating rewiring of Ephrin signaling during the development of resistance. Additionally, 15 out of the 37 differentially expressed proteins were associated with p53.
- DigiWest protein profiling detected expression differences for approximately 200 proteins from mammary carcinoma tissue that was collected via laser capture microdissection. Additionally, Her2 signal intensity detected via histology was reproduced by the DigiWest.
Now that Templin’s group has demonstrated proof of concept with DigiWest, they are working to broaden its applicability. While the primary application of their method thus far has been biomarker discovery and lead compound profiling in oncology research, DigiWest’s ability to simultaneously analyze up to 600 proteins, including phospho-status, would likely also be of interest to preclinical and clinical scientists in other fields. They have also been working to expand their antibody panels from human and mouse to also include rat, dog, and mini pig as well.
Want to learn more?
Read the Nature Communications article.
Visit the DigiWest website.
DigiWest is a trademark of NMI registered in Germany.
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