Quench HRP with sodium azide - (Nov/13/2008 )
Hello Everyone,
I would like to know if anyone has a protocol for quenching HRP (conjugated to a secondary antibody) with sodium azide. I am going to probe my blot with an primary antibody made in mouse, then use a goat anti-mouse secondary conjugated to HRP. I would then like to quench the HRP so I can re-probe my membrane (with a primary Ab directly conjugated to HRP) without having to strip it.
My current plan of action:
Develop first blot
Wash membrane in 0.1% Sodium Azide in TBST for 10 min (3 washes)
Wash membrane in TBST for 30 min (change wash every 5 min)
Add chemiluminescent substrate to membrane to see if HRP is effectively quenched
I am not sure if I am using enough sodium azide or if I am washing enough after adding sodium azide. Any input would be appreciated!
Thanks!
Won't the azide just wash off? I would have thought that the quenching would not be a permanent effect unless the azide changes the hrp in some way.
I would like to know if anyone has a protocol for quenching HRP (conjugated to a secondary antibody) with sodium azide. I am going to probe my blot with an primary antibody made in mouse, then use a goat anti-mouse secondary conjugated to HRP. I would then like to quench the HRP so I can re-probe my membrane (with a primary Ab directly conjugated to HRP) without having to strip it.
My current plan of action:
Develop first blot
Wash membrane in 0.1% Sodium Azide in TBST for 10 min (3 washes)
Wash membrane in TBST for 30 min (change wash every 5 min)
Add chemiluminescent substrate to membrane to see if HRP is effectively quenched
I am not sure if I am using enough sodium azide or if I am washing enough after adding sodium azide. Any input would be appreciated!
Thanks!
you need to get rid of primary and seconday abs before doing another primary secondary. There will be too much considerations or interference otherwise. Just strip.
I would like to know if anyone has a protocol for quenching HRP (conjugated to a secondary antibody) with sodium azide. I am going to probe my blot with an primary antibody made in mouse, then use a goat anti-mouse secondary conjugated to HRP. I would then like to quench the HRP so I can re-probe my membrane (with a primary Ab directly conjugated to HRP) without having to strip it.
My current plan of action:
Develop first blot
Wash membrane in 0.1% Sodium Azide in TBST for 10 min (3 washes)
Wash membrane in TBST for 30 min (change wash every 5 min)
Add chemiluminescent substrate to membrane to see if HRP is effectively quenched
I am not sure if I am using enough sodium azide or if I am washing enough after adding sodium azide. Any input would be appreciated!
Thanks!
alternatively incubate the blot with 3% H2O2 for 15 min, then wash...