I saw some other people presenting similar problems.
For a few years now, we were using Dynabeads M280, coupling them to a biotinylated antibody to perform immunopurification of target molecules. All of a sudden, after opening a new lot of magnetic beads, the beads do not seem to work anymore, and all of the biotinylated antibody can be detected in the unbound fraction by indirect ELISA.
We did not change anything in the protocol and checked the pH of all buffers already. The problem presents itself with all the different biotinylated antibodies that we checked, all of which are from the same lot or even from the same aliquotes that previously worked well.
We contacted the company, and tried other beads lots, as well as the magnetic beads from a competitor.
The way I see it, the binding step between biotin and streptavidin is clearly the problem, and we tried different conditions (time, temperature, rotation vs agitation) in order to improve it, but nothing seems to work anymore.
I was wondering if someone is still using such beads successfully, or has ever encountered any problems of this kind.
Thanks in advance!
Are you using biotin alone (like Biotin-OSu / Biotin NHS-ester) or biotin with a spacer (like Biotin-X-Osu) for labeling your antibodies?
Are you using a new blocking agent that might contain free biotin that would compete with the antibodies?
Thank you for the help.
Wolfgang Schechinger we are using commercial antibodies, and unfortunately I cannot find such information on the website of the relative companies. What could be a potential problem involving spacers?
Adam B Shapiro No, we are not using any blocking agent on this step. The beads themselves come suspended in a buffer containing 0.1% BSA, but that has always been the case. We even tried to wash multiple times the beads in order to remove any potential unbound contaminant but that did not affect the results.
@Fabio, when no spacer is used, biotin does not always fit well into the binding pockets of streptavidin.
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