Identified issues and solutions will be listed here.
Issue 1:
Biotinylation of the spacer proteins is inefficient.
Solution 1:
The protocol described in the paper, established at Rockefeller University, did not work at Fred Hutch because the MilliQ at the Hutch is under oxidizing conditions. Using a low concentration of TECP resolved the issue (see the step-by-step protocol above).
Issue 2:
The MagIC-cryo-EM beads do not capture the target protein.
Solution 2:
It is possible that the beads are not assembled as intended. We highly recommend performing SDS-PAGE at each step of the bead preparation process, including for the streptavidin beads, prior to protein assembly. In particular, non-specific binding of the target-capturing module can reduce the efficiency of the pull-down.