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An analysis of critical factors for quantitative immunoblotting

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Sci. Signal.  07 Apr 2015:
Vol. 8, Issue 371, pp. rs2
DOI: 10.1126/scisignal.2005966

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Best practices for immunoblotting

Immunoblotting, also known as Western blotting, is a common technique used by cell and molecular biologists to detect the presence of proteins, changes in protein abundance, and the presence and changes in abundance of posttranslational modifications. Unfortunately, many factors influence reproducibility and the ability to produce quantitative results with this technique. Janes provides a detailed analysis of how variations in sample preparation, detection scheme, and normalization approach affect the reliability, reproducibility, and quantitative value of proteins evaluated by immunoblotting.

Abstract

Immunoblotting (also known as Western blotting) combined with digital image analysis can be a reliable method for analyzing the abundance of proteins and protein modifications, but not every immunoblot-analysis combination produces an accurate result. I illustrate how sample preparation, protocol implementation, detection scheme, and normalization approach profoundly affect the quantitative performance of immunoblotting. This study implemented diagnostic experiments that assess an immunoblot-analysis workflow for accuracy and precision. The results showed that ignoring such diagnostics can lead to pseudoquantitative immunoblot data that markedly overestimate or underestimate true differences in protein abundance.

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