I recently read that using sodium azide to preserve the HRP-conjugated secondary antibodies in their blocking buffer will inactive the HRP-conjugation. Is there another preservative that I can add to the solution instead that will not inactivate the HRP?
Usually secondary antibodies are prepared fresh for each use and are not reused. It would also be a little dangerous to reuse secondary antibodies as you would be introducing variables into your experiments that may cost you more time, effort and money to fix than preparing fresh antibodies each time.
Primary antibodies differ in that respect for very highly expressed proteins or if your antibody is very good, the dilution factor suggested would probably be way over what is actually required to get good detection. In that case you can probably get away with using primaries two to three times when diluted in 5% BSA with sodium azide and refrigerated during incubation and storage.
You don't need to add azide in blocking buffer, If you perform secondary antibody incubation at 4 degree C. This can be reused for at least one week without any apparent contamination. But, I personally wouldn't reuse diluted secondary antibody.
The point of azid is to prevent microbial growth on the antibody, so any anti-microbial should do. Nevertheless I agree with Sridevi Mony that recycling seconndary antibodies is probebly a bad idea.
Yoram
You can reuse diluted primary antibody specially if it was diluted in BSA. Using milk can be a little more problematic, but it can still be done. You need to see if the reactivity is still good for your specific antibody. I would use Benzalkonium Chloride as a preservative. Farhad
Hi Jessica,
there is an excellent commercial product from Seramun: http://www.seramun.com/products/stabilizers/stabilizers-for-hrp-conjugates.html, They are using Kathon as preservative.
I use LiCor secondary antibodies and sodium azide is not a problem. The antibodies are reusable; the imaging process is easy and the dynamic range is high.
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