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I have done some Western blots where I have analysed the expression levels of a certain protein in brain homogenates from wild-type and knockout mice. When preparing brain homogenates, I was advised to prepare them in pairs, so that on the same day two brain homogenates, one from a wild-type mouse and the other from a knockout mouse were prepared.

I know that paired analysis is useful for comparing differences visually on the blot between samples from two experimental groups. However, I was wondering whether there are any other benefits (e.g. for statistical analysis) to preparing samples in pairs for a Western blot analysis besides just ease of visual comparison? Any insights are appreciated.

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This point is not unique to Western Blot, but to any case-control study: if there is variability inherent in the preparation procedure, this variability will be reduced when the two samples are prepared simultaneously. This is perhaps easier understood using an analogy: if a toaster fits two toasts, but there is variation in the toasting time - the toasts that were in the toaster together are likely to be cooked/burned to the same level.

Statistically, one takes the full advantage of such improvements when using the statistical techniques specifically designed for paired data, such as paired difference test (see also paired samples t-test). But even unpaired statistical methods are likely to show better performance (in terms of identifying significant results).

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