| BackgroundIn Western blotting, a suitable loading control is indispensable forcorrecting errors in the total amount of loaded protein. Immunodetection ofhousekeeping proteins and total protein staining have traditionally been usedas loading control methods. Direct Blue71(DB71) staining–a novel,sensitive, dye-binding staining method compatible with immunodetection–may offer advantages over these traditional loading control methods.MethodsThree common neuroscientific samples (human plasma, humanoligodendrocytes, and rat brain) were employed to assess DB71staining as aloading control method for Western blotting. DB71, CBB, one traditionalhousekeeping protein, and one protein of interest were comparativelyassessed for reliability and repeatability and linear dynamic range over2.5-40μg of protein loaded. DB71’s effect on the reliability andrepeatability and linear dynamic range of immunoreaction were also assessed.ResultsAcross all three sample types, DB71was either equivalent or superiorto CBB and housekeeping protein-based methods in terms of reliability andrepeatability and linear dynamic range. Across all three sample types, DB71staining did not impair the reliability and repeatability or linear dynamicrange of immunoreaction.ConclusionDB71staining can be used as a destaining-free alternative loadingcontrol method for Western blotting. |
