Hi,
I'm trying to stimulate human T cells with magnetic beads coated with antibodies. I have streptavidin-coated beads as well as biotinylated antibodies. According to the vendor, they usually incubate cells with antibodies first and then mix them with beads. I'm wondering if it's feasible to immobilize the antibodies onto beads first and then add to cells. Thanks!
I think this would work as long as (1) you are able to keep the beads in suspension by stirring the culture medium, and (2) biotin in the medium does not cause the antibodies to dissociate from the beads by competition.
The Biotin-Streptavidin interaction is one of the strongest non-convalent interactions known to science. I would not expect that free biotin is able to dissociate the complexes. That's why desthio-biotin and other reagents have been developed, where the interaction with streptavidin is weaker and dissociation indeed may be achieved by adding biotin.
The potential problem that I can see is free biotin (e.g. bound to albumin) in the culture medium, that will happily and irreversibly cover all the binding sites of the streptavidin and outcompete the antibodies. Biotin-free BSA is available to make up serum free media, but quite expensive.
That's why I'd react the antibodies first with the streptavidin beads, but it's easy (and reasonable) to investigate and possibly to demonstrate that the order really matters.
If you use NHS-Biotin (or similar) to label the antibodies, you might get low binding to strep, as this handle is frequently too short for the biotin to reach the binding pocket. Biotin-C6-NHS (AKA Biotin-X-Osu) with an aminohexanoic acid spacer facilitates the binding to streptavidin. For more sophisticated and longer spacers (hydrophilic poly-ethyleneglycoles, poly-sarcosins, etc., you may find useful information in this brochure by Iris Biotech: https://www.iris-biotech.de/global/biotinylation-reagents
Kun Yun I meant that if you add the beads to the cell culture, they will settle to the bottom if there is no stirring or agitation. Some types of magnetic beads are very dense and will drop to the bottom of the container very quickly.
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