RAD18_RAD6_MAGEA4_Cross-linking_Mass_Spectrometry

RAD18 and RAD6 were co-expressed in E.coli, RAD18 harbors an N-terminal 6His tag which was utilized for affinity chromatography via cobalt affinity resin. The complex was then subjected to size exclusion chromatography. 5 X molar excess MAGEA4 (also has N-terminal His-tag) was added to RAD18-RAD6. The complex was crosslinked with 0.5 x Molar BS3 to lysine residues within the complex for 10 minutes at room temperature, shaking at 600 rpm. The reaction was quenched with 100 mM Tris pH 8, incubated at 35 °C for 10 minutes, shaking at 600 rpm.