Azide interaction with HRP... - (Aug/10/2009 )
Hello, i recently bought an antibody (p21) wich is in azide solution (as conservation agent). Since i use this Ab, I always get bad results in western blot... So i would like to know if it could be possible that azide could interact badly with HRP. (enzyme bind with the secondary antibody). Meaning interfere with first and second antibodies binding or causing degradation of western revelation solution... (i do remember something about interaction between azide and HRP from university, but i'm really not sure)
Thanks a lot for your help!
HRP is an enzyme that will be deactivated/inhibited by azide
Azide can kill HRP
You can remove the azide by dialysis or centrifugal filtratiom (such as an amicon filter)
First try increasing the washes between your primary and secondary antibody. Add more volume and increase the amount of tween.
Have you tested PBS vs TBS for your buffers? Some antibodies are very particular!
If this doesn't help, you may want to try azide removal. Centrifugal filtration works best for small volumes and dialysis for large volmes.
NaN3 (usually 0.1%) is often found in antibody buffers. It is inside the buffer to prevent bacterial contamination. Unless you buy a hybridoma line and make your own antibodies, it will usually be there when you purchase it from companies. The concentration of NaN3 is very little to begin with, and after your subsequent washes after antibody incubation... it is negligible. The resultant concentration of NaN3 after 3 washes is essentially nil and will not interfere with HRP.
if you skimp on the wash, you may not wash out the azide. Washing should be very generous (lots and lots of wash buffer)
i'm gonna try this