Hello everyone,
I am currently working on assessing the interaction between biotinylated fluorescent silica beads (diameter is about 200nm) and free streptavidin-agarose beads in solution. The problem I encounter is the purification of my streptavidin-agarose once the biotinylated beads are attached on their surface : indeed, there are still a lot of unbound fluorescent silica beads in my solution and I cannot find a way to isolate the streptavidin-agarose beads from them. I've been suggested to purify by centrifugating in a 20% sucrose solution, in order to collect the streptavidin-agarose in the supernatant, but everything ended up in the pellet. Would an increase in sucrose percentage help ? Do you have any protocol or suggestion please ?
Thanks a lot.
You probably need to increase the density of the medium. Try using a gradient instead of a single concentration in order to figure out what % sucrose will separate the two materials. For example, 25-60% sucrose. If one type of beads ends up pelleting at 60% but the other type ends up at 45%, then you know that you can use a concentration in between for separating them in the future.
could you do it with magnetic beads and separate with a magnet ? Alternativey filter the beads through a .45 micron filter and collect the supernatant
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