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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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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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