I used to make streptavidin agarose myself, which had double the binding capacity of the commercial materials. I used cyanogen bromide to activate Sepharose 6 FF. In your case with magnetic beads you could follow the protocol attached from Chemicell, where you should be able to be able to increase the binding capacity. Look up their website, they are "local" to you being a German company.

Stanley Ng is right. Unless your beads are provided pre-derivatized you'll need to activate them.

HOWEVER, magnetic beads usually *are* provided pre-derivatized, either with COOH or (more often) NH2. If you already have the beads, let us know what you've got. Otherwise I recommend you buy beads that come with a protocol. For NH2 beads, you'll typically use NHS as a link.

Thank you so much for the protocol and ideas. I was thinking to oxidize the KH moiety of HRP with periodate and then couple to NH2-beads. Does this make sense?

What are you trying to do here, what is your end goal? why would you cross-link HRP to beads? Are you using Strep-HRP?

Dear Stanley, sorry of course Streptavidin-HRP.

We want to enrich circulating melanoma cells from blood samples. To this end, we have produced biotinylated scFvs at the C terminus that bind specifically to the surface of melanoma cells, which we have confirmed by flow cytometry.

These scFvs will be coupled via biotin - streptavidin/anti-biotin interaction with magnetic particles to magnetically isolate the cells.

The HRP is really superfluous to your experiment then.

If you are looking to isolate the cells and recover, then using monomeric avidin would be the best method, it has the selectivity of strep, but has very gentle elution conditions (2mM biotin in whatever buffer you want), you can then strip the beads and reuse with the same sample until you've recovered as many cells as you need.

https://divbio.eu/en/suppliers/bioclone-inc/262680-bcmag-tm-monomer-avidin-magnetic-beads-2-ml-beads-kit