Gelcode blue

Probe labeled and rhodamine appended proteins were resolved via SDS-PAGE. Ten μg protein was added to each well of a 4–12% Bis-Tris gel (Invitrogen). Samples were run at 200 V, 35 mA for 45 min. Gels were imaged using a Typhoon FLA 9500 (General Electric) followed by total protein staining and imaging using GelCode Blue and GelDocEZ (BioRad Laboratories).

HEK293 cells, stably overexpressing HA-MRP4 were seeded on ten 100 mm dishes and grown until completely confluent. After lysis, the cell lysate was mixed with anti-HA beads overnight at 4°C for immunoprecipitating HA-MRP4. The beads were washed and the bead-bound MRP4 was eluted with 100 mM glycine (pH 2.2) and neutralized with 150 mM tris (pH 8.8). The eluted MRP4-containing macromolecular complex was run by SDS-PAGE on 4–15% gradient gels (Bio-Rad, Hercules, CA) and visualized with GelCode Blue (manufacturer’s information or add to reagents section). The MRP4-containing macromolecular complex was subjected to mass-spectrometric analysis for identifying the MRP4-interacting partners [22 (link)].