Using NaN3 to quench HRP conjugated secondary Ab (View forum version)



MaggieRoara

Posted 11 June 2009 - 02:11 AM

Hi everyone,
Can anyone tell me if they have used NaN3 to kill HRP conjugated secondary Ab before reprobing. My reprobes are from a different animal source. I dont want to have protein loss because of stripping

krisztina

Posted 15 July 2009 - 02:42 PM

Hi,

Yes, I've done that routinely and had no problems with re-probing (sometimes ;) ).
I usually wash the membrane 2X after ECL and block in milk for 1 hr RT. When I am concerned (strong signal from previous detection) then I add azide to the blocking solution as well (up to 5X than in the primary ab solution used to conserve the ab, usually 1X azide is OK).
But most of the times I just re-use a primary ab aliquot that already has azide in it and it is OK. Especially if the 2nd ab-s are from different animals.
However, I always use 2nd ab-s at the lowest possible concentration, that can also be a reason why the above conditions usually work fine.

Good luck,
Krisztina


Hi everyone,
Can anyone tell me if they have used NaN3 to kill HRP conjugated secondary Ab before reprobing. My reprobes are from a different animal source. I dont want to have protein loss because of stripping