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Article Dans Une Revue Analytical Biochemistry Année : 2009

Multiple reprobing of Western blots after inactivation of peroxidase activity by its substrate, hydrogen peroxide

Stéphane Charpentier
Thierry Normand
Cedric Sarre
Lucile M. Mollet

Résumé

Sequential detections of different proteins on Western blot save time and precious samples. The main problem concerning reprobing is that stripping buffers can unbind both the antibody and the tested antigen. An original reprobing method has been set up based on horseradish peroxidase (HRP) inhibition after enhanced chemiluminescence detection. Instead of removing previously fixed antibodies as common stripping buffers do, the HRP activity linked to the secondary antibody is irreversibly inhibited by excess of hydrogen peroxide. A 15-min incubation allows one to perform at least five different sequential detections without losing significant amounts of blotted proteins.

Dates et versions

hal-00525339 , version 1 (11-10-2010)

Identifiants

Citer

Alexis D. Sennepin, Stéphane Charpentier, Thierry Normand, Cedric Sarre, Alain Legrand, et al.. Multiple reprobing of Western blots after inactivation of peroxidase activity by its substrate, hydrogen peroxide. Analytical Biochemistry, 2009, 393 (1), pp.129-131. ⟨10.1016/j.ab.2009.06.004⟩. ⟨hal-00525339⟩
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