Dear colleagues,

I am seeking advice on troubleshooting a challenging in vitro supercoiling assay. My goal is to demonstrate the histone chaperone activity of a recombinant protein by observing the supercoiling of a relaxed plasmid.

I have run the assay several times but consistently encounter a specific issue: the reaction appears to stall.

Has anyone encountered a similar "stalled" or "ceiling" effect in this type of assay?

Is my molecular chaperone inactive? However, I've tested the activity using the method described in the literature and found no issues. The histone octamer was prepared by in vitro dialysis after purification of H2A/H2B/H3/H4, and SDS-PAGE analysis showed no issues.