Summary
Nonspecific interactions between blotted proteins and unrelated secondary antibodies generate false positives in immunoblotting techniques. Some procedures have been developed to reduce this adsorption but they may work in specific applications and be ineffective in other ones. “Double-blotting” has been developed to overcome this problem. It consists of interpolating a second blotting step between the usual probings of the blot membrane with the primary antibody and the secondary antibodies. This step, by isolating the primary antibody from the interfering proteins, guarantees the specificity of the probing with the secondary antibody. This method has been developed for the study of erythropoietin in concentrated urine since a strong nonspecific binding of biotinylated secondary antibodies to some urinary proteins is observed using classical immunoblotting protocols. However, its concept makes it usable in other applications that come up against this kind of problem. This method is expected to be especially useful for investigating proteins that are present in minute amounts in complex biological media.
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Acknowledgments
The DB process has been patented (2 786 273) by “Hospices Civils de Lyon”, and by “Laboratoire National de Dépistage du Dopage”with “Hospices Civils de Lyon” as PCT/FR01/01331.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Lasne, F. (2009). Double-Blotting: A Solution to the Problem of Nonspecific Binding of Secondary Antibodies in Immunoblotting Procedures. In: Kurien, B., Scofield, R. (eds) Protein Blotting and Detection. Methods in Molecular Biology, vol 536. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-542-8_23
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DOI: https://doi.org/10.1007/978-1-59745-542-8_23
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-934115-73-2
Online ISBN: 978-1-59745-542-8
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