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Dot blot is a straightforward method for detecting biological samples, such as proteins or nucleic acids. It shares principles with western and southern blotting but differs as samples are applied directly to a membrane instead of being transferred from a gel. Due to its simplicity, dot blot is versatile and supports various applications, including monitoring labeling efficiency with probes, estimating protein concentration, and comparing antibody performance. For instance, to assess labeling efficiency with biotin, one would compare a labeled sample to a known biotinylated reference standard using serial dilutions. A 10-fold dilution is recommended for a broad range of samples.