Primary antibody and Sodium azide
Posted 10 August 2009 - 09:49 PM
Days a few back I faced problems in getting signals from my blot, while I diluted the primary ab in 3% BSA and with 0.1% NaN3. While diluting the same ab with similar condition, only reducing the NaN3 to 0.01% resulted the clear signal in the band.
Does any one know if NaN3 has a deleterious effect on the primary an other than being an anti-bacterial preservative?
Posted 11 August 2009 - 04:02 AM
Posted 11 August 2009 - 07:04 AM
Were you using an HRP-based detection? NaN3 (azide) kills HRP (there is another discussion on this in the immuno forum), you may have diluted the azide enough to wash most of it out so it doesn't kill the detection system
the dedection is based on HRP. I do three washes with TBS-T, 5 min each before adding HRP-conjugated secondary ab. Probably the washes are not enough. I'll check increasing the wash.