Several additives can be added to antibody’s stock and working solutions:
- Sodium Azide (NaN3) – preservative that usually added to a final concentration of 0.01-0.02%. It should be noted, though, that this addition should be avoided in case the antibody will be used on live cell samples or when using a HRP-conjugated antibody (inhibitory effect).
- BSA – When using low concentration antibody stock the BSA can serve as a crowding effect agent.
- Serum - Serum can be used when Milk or BSA are not efficiently blocking cross-reactivity of the antibody used. When using serum, you should make sure the serum’s origin differs from that of the antibody.
- Non-fat Milk – When performing western blotting it is recommended to dilute the antibody into a 5% milk solution based on Phosphate Buffer Saline (PBS). Milk spoils quickly thus it is possible (and recommended) to add Sodium Azide (see above) to the solution.
- Detergents (Tween-20/NP-40/SDS) – Detergents can be added to the working solution (specifically, western blotting application) as it can increase the signal-to-noise ratio due to elimination of non-specific binding of the antibody’s Fc region with various proteins in the sample and blocking solution. Usually 0.05% of Tween-20 should do the trick but sometimes antibodies can be quite sticky thus it is possible to switch to a different detergent. Remember that protein’s can denature in the presence of detergent’s micelles thus use detergents with care (first use NP-40 and only then SDS).
Expedite Your Western Blots and ELISA washes
Even though ELISA and western blotting protocols state that you should wash your membrane/wells three times for 15 minutes after admission of antibody solution, it’s actually better to do several large volume washes, each for a short period of time (no more than a 30 seconds) and then do a final wash for 10 minutes. Basically it will both save you time and in addition, the washes with large volume will remove most of the antibody solution quicker.
Chen Guttman is a Graduate Student at the Zarivach lab in Ben Gurion University. Chen blogs at benchwise and serves as BioData's community liason.