Multiple reprobing of Western blots after inactivation of peroxidase activity by its substrate, hydrogen peroxide.
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.
Sennepin
Alexis D
AD
Centre de Biophysique Moléculaire, CNRS UPR4301, affiliated with Université d'Orléans, 45071 Orléans, France.
Charpentier
Stéphane
S
Normand
Thierry
T
Sarré
Cédric
C
Legrand
Alain
A
Mollet
Lucile M
LM
D031215:Armoracia / Q000201:enzymology; D015153:Blotting, Western / Q000379:methods*; D004789:Enzyme Activation; D006735:Horseradish Peroxidase / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D015335:Molecular Probes / Q000032:analysis*; D013379:Substrate Specificity
D015335:Molecular Probes; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase