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Direct B lue 71 staining as a destaining‐free alternative loading control method for W estern blotting
Author(s) -
Zeng Li,
Guo Jing,
Xu HongBo,
Huang Rongzhong,
Shao Weihua,
Yang Liu,
Wang Mingju,
Chen Jianjun,
Xie Peng
Publication year - 2013
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201300140
Subject(s) - repeatability , staining , blot , housekeeping , reliability (semiconductor) , chromatography , chemistry , biology , biochemistry , genetics , power (physics) , physics , quantum mechanics , gene
In W estern blotting, a suitable loading control is indispensable for correcting errors in the total amount of loaded protein. Immunodetection of housekeeping proteins and total protein staining have traditionally been used as loading control methods. Direct B lue 71 ( DB 71) staining—a novel, sensitive, dye‐binding staining method compatible with immunodetection—may offer advantages over these traditional loading control methods. Three common neuroscientific samples (human plasma, human oligodendrocytes, and rat brain) were employed to assess DB 71 staining as a loading control method for Western blotting. DB 71, CBB , one traditional housekeeping protein, and one protein of interest were comparatively assessed for reliability and repeatability and linear dynamic range over 2.5–40 μg of protein loaded. DB 71's effect on the reliability and repeatability and linear dynamic range of immunoreaction were also assessed. Across all three sample types, DB 71 was either equivalent or superior to CBB and housekeeping protein‐based methods in terms of reliability and repeatability and linear dynamic range. Across all three sample types, DB 71 staining did not impair the reliability and repeatability or linear dynamic range of immunoreaction. Our results demonstrate that the DB 71 staining can be used as a destaining‐free alternative loading control method for W estern blotting.