Dear Mark,

Avid in is known to be inactivated by oxidizing agents such as ozone, peroxide or strong light source. Please refer to the following literature:

Green, N.M., Biochem. J., 89, 599 (1963). "Avidin. 3. The nature of the biotin-binding site."

cheers,

What I am wondering is, why inactivate the biotin instead of just leaving it out?

In any case, add the streptavidin from a 1 mg/ml  (~75 µM monomer) stock solution to the medium containing diluted biotin, rather than to the biotin stock solution. This will require much less streptavidin.

If you are using about 1 nM (10-9 M) biotin in the medium, as is typical, you could use 1 nM streptavidin to bind it all up, since the dissociation constant for the binding interaction is 10-14 M. To be on the safe side, allowing for measurement error, add twice as much streptavidin (as monomer) as biotin.

Thanks a lot, Adam! The idea to add streptavidin is to check if there's an "immediate" effect of biotin inactivation, while that's true - if you leave it out it will exhaust "naturally".. 

We just received that streptavidin tube (Promega #Z7041) and there's 1mg of the powder. Should I dissolve it in media (how it must be kept after that... +4? ) or in PBS/water? Then I could aliquot and freeze it at -20.. What's the best solution?

Thanks again!

Since you will be diluting the stock solution substantially, you can make it up in any convenient buffer. You should be able to flash-freeze it if you want to store it long-term at -80oC. The only thing I would recommend against if you plan to freeze it is to dissolve it in PBS or other phosphate-based buffer.

Also, if you are going to add it to cells in culture, it will have to be sterilized by filtration. The risk there is that the concentration may decrease if some of it gets stuck on the filter. You should measure the concentration before and after filtration.

You said there is 1 mg of powder. Is it all streptavidin, or are there other substances present? You should calculate the concentration based on the amount of streptavidin protein, not the amount of powder.

Thank you, Adam.

It's all liophylized streptavidin.

The only problem is that we use 30µM of biotin per well for our experiments, while the initial concentration of streptavidin is about 75µM (as you mentioned before, when you dilute 1mg in 1ml)..! The stock of biotin we have is 100x - which is 3mM. (to add 10µl per 1ml).

Do you really need 30 uM biotin in the wells? This seems extremely high.

Unfortunately, yes. That's what paper says... http://www.ncbi.nlm.nih.gov/pubmed/22406856