I have a His-tagged recombinant protein that I'm attaching to Dynabeads for aptamer targeting. Once the aptamer library binds, how do I remove the aptamers from the protein for amplification? I've seen others use 95C heat, urea or high salt. I've also seen imidazole to remove the whole protein-DNA complex from the beads and put that into PCR.
Is there a way to reuse the same protein (either by keeping it attached to the beads or reattaching it if it's eluted) or would the protein structure be disturbed in these aptamer elution processes?
Hi Megan,
usually, we are immobilizing a new batch of His-tagged protein for each SELEX round. We prefer to elute candidates though heating, which of course should unfold the protein.
You're right that a change in pH or salt concentrations could allow you to reuse the beads. But then, would you be sure that no candidates from the previous round are still on the protein?
You need only 5-20 pmoles of protein per round. If your target is not too expensive, i would immobilize a new set for each round
I totally agree with heat approach, because no additional chemicals would be introduced. I sometimes thinking about that, by the way, how about adopting and introducing an antibody into solution to carry out a competitive binding? If it happened, the ssDNA/RNA would be released into solution, like an exchange. However, it just a hypothesis.
Peng Xiao Meral Yüce
Laurent Azéma Thank you so much for your answers! Do you just heat 95C 5 mins? And then you put that solution directly into PCR?I have used the carboxylic acid beads before for protein immobilization, but this is a recombinant his-tagged protein that I want to keep in that conformation to mimic its expression on a cell surface.
usually 5mn, 75°C. But remember that the protein used for the selex round will have to be discarded. You will have to prepare a new batch of Ni-NTA beads/protein for the next round.
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