For in vitro binding assays, Myc or FLAG-tagged GFP, MAD1L1, MAD2L1, or
ELYS (N-terminal fragment) were in vitro transcribed and translated (IVT)
using TNT Quick Coupled Transcription/Translation System (Promega, Madison, WI) in 10
μL reactions. Myc beads (MBL, Sunnyvale, CA) were washed three times and
equilibrated with wash buffer (50 mM Tris pH 7.4, 200 mM KCl, 1 mM DTT, 0.5% NP-40, and
Halt Protease and Phosphatase Inhibitor Cocktail). IVT reactions were added to
equilibrated Myc beads and incubated for 1.5 h at 30 °C with gentle shaking, and
after binding, beads were washed three times with wash buffer and eluted by boiling for 10
min with 2× Laemmli SDS sample buffer. The samples were then resolved using a
4–20% gradient Tris gel with Tris-Glycine SDS running buffer, transferred to an
Immobilon poly(vinylidene difluoride) (PVDF) membrane (EMD Millipore, Burlington, MA), and
the membranes were analyzed using a PharosFX Plus molecular imaging system (Bio-Rad,
Hercules, CA).
ELYS (N-terminal fragment) were in vitro transcribed and translated (IVT)
using TNT Quick Coupled Transcription/Translation System (Promega, Madison, WI) in 10
μL reactions. Myc beads (MBL, Sunnyvale, CA) were washed three times and
equilibrated with wash buffer (50 mM Tris pH 7.4, 200 mM KCl, 1 mM DTT, 0.5% NP-40, and
Halt Protease and Phosphatase Inhibitor Cocktail). IVT reactions were added to
equilibrated Myc beads and incubated for 1.5 h at 30 °C with gentle shaking, and
after binding, beads were washed three times with wash buffer and eluted by boiling for 10
min with 2× Laemmli SDS sample buffer. The samples were then resolved using a
4–20% gradient Tris gel with Tris-Glycine SDS running buffer, transferred to an
Immobilon poly(vinylidene difluoride) (PVDF) membrane (EMD Millipore, Burlington, MA), and
the membranes were analyzed using a PharosFX Plus molecular imaging system (Bio-Rad,
Hercules, CA).