The feasibility of this method was tested here at both cellular and tissue levels. In order to achieve this goal, we have introduced a novel multi-unit plate in our analysis ( Figure 1A), and named this method as Quantitative Dot Blot analysis (QDB), and the plate as QDB plate. We reasoned that achieving direct quantification of individual dots in the traditional dot blot analysis, rather than through an extra image conversion process, should significantly improve upon the traditional method. We have now developed a novel immunoblot method as convenient and robust as the traditional dot blot analysis, yet in high throughput format to meet this demand. Meanwhile, with the rapid development of effective high-throughput tools in genetic research, there are strong demands for complementary high throughput immunoblot methods on a daily basis for biomarker identification and other association studies at protein level. Nonetheless, the applications of these techniques are still limited in basic research lab for various reasons, including limited availability for prefabricated ELISA kits and lack of easy access to RPPMs. In fact, both enzyme-linked immunosorbent assay (ELISA) and reverse phase protein microarray (RPPM) can be considered as Dot Blot analysis in a high throughput format.
As an alternative, Dot blot analysis was developed to simplify the process of Western blot analysis.
However, its limitations, including complicated processing steps, limited ability to process many samples, ambiguity in result analysis and requirement of large amount of total protein lysate for analysis, determines this technique an unlike choice for high throughput analysis. Until now, Western blot analysis remains the most commonly used immunoblot tool in the basic research lab almost 40 years since its invention. Lacking of an accessible high throughput immun-oblot method significantly hinders any attempts to investigate the molecular basis of biological and pathological processes systematically in an average research lab. We believe that the adoption of QDB analysis would have immediate impact on biological and biomedical research to provide much needed high-throughput information at protein level in this “Big Data” era. Using QDB technique, we were able to observed an age-dependent significant alteration of CAPG protein expression level in TRAMP mice. This method was evaluated at both cellular and tissue levels with unexpected observations otherwise would be hard to achieve using conventional immunoblot methods like Western blot analysis. In addition, the small amount of sample lysates needed for analysis means significant saving in research sources and efforts. Its convenience in analyzing large number of samples also enables bench scientists to examine protein expression levels from multiple parameters. With the defined linear range, QDB analysis fundamentally transforms traditional immunoblot method into a true quantitative assay. We proposed here Quantitative Dot Blot analysis (QDB) to meet this demand. Lacking access to an affordable method of high throughput immunoblot analysis for daily use remains a big challenge for scientists worldwide. Received: FebruAccepted: MaPublished: April 19, 2017 Keywords: quantitative dot blot, high throughput, proteomics, immunoblot analysis, western blot *These authors have contributed equally to this work ChinaģDepartment of Chemistry, BMC, Uppsala University, Uppsala, Sweden Geng Tian 1, *, Fangrong Tang 2, *, Chunhua Yang 1, Wenfeng Zhang 2, Jonas Bergquist 3, Bin Wang 1, Jia Mi 1, 3 and Jiandi Zhang 1, 2ġMedicine and Pharmacy Research Center, Binzhou Medical University, Yantai, P.
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