  Single-molecule F\"orster resonance energy transfer (smFRET) of molecular
motors provides transformative insights into their dynamics and conformational
changes both at high temporal and spatial resolution simultaneously. However, a
key challenge of such FRET investigations is to observe a molecule in action
for long enough without restricting its natural function. The Anti-Brownian
ELectrokinetic Trap (ABEL trap) sets out to combine smFRET with molecular
confinement to enable observation times of up to several seconds while removing
any requirement of tethered surface attachment of the molecule in question. In
addition, the ABEL trap's inherent ability to selectively capture FRET active
molecules accelerates the data acquisition process. Here we exemplify the
capabilities of the ABEL trap in performing extended timescale smFRET
measurements on the molecular motor Rep, which is crucial for removing protein
blocks ahead of the advancing DNA replication machinery and for restarting
stalled DNA replication. We are able to monitor single Rep molecules up to 6 s
with 1 ms time resolution capturing multiple conformational switching events
during the observation time. Here we provide a step-by-step guide for the
rational design, construction and implementation of the ABEL trap for smFRET
detection of Rep in vitro. We include details of how to model the electric
potential at the trap site and use Hidden Markov analysis of the smFRET
trajectories.
