Abstract
Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.
Highlights
Alongside with the exciting advancements in genomic research in the recent years, biomedical research field witnesses the significant advancement in proteomic research
We demonstrated the process of quantitative dot blot analysis (QDB) analysis to achieve absolute determination of CAPG protein level in mouse tissues including kidney, spleen, and prostate
Compared to Enzyme linked immunosorbent assay (ELISA), QDB analysis requires minimum efforts to be developed in a regular lab
Summary
Alongside with the exciting advancements in genomic research in the recent years, biomedical research field witnesses the significant advancement in proteomic research. Enzyme linked immunosorbent assay (ELISA)[5,6,7] and reverse phase protein microarray (RPPM)[8,9] can be considered the high throughput format of immunoblot analysis All these immunoassay methods, except ELISA, measure the relative expression level of a specific protein. We present a detailed protocol for QDB analysis and demonstrate the method by determining the absolute protein content of a specific protein, CAPG, in three different mouse tissues including kidney, spleen, and prostate. We think that this detailed protocol well illustrates the feasibility and convenience of this method, and provide guidance on how to avoid the potential pitfalls in the practice of this method.
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