Lysis Buffer (10mL). Combine 5mL 1x HNG, 1 PI tablet, 1mL of 1% NP40, and 50µL of magnesium chloride.
Glutathione sepharose beads
Glass beads
Protocol
Inoculate cells in appropriate media overnight.
Re-suspend cells in 1mL of lysis buffer.
Lyse cells: place approximately 1mL of glass beads into sonicator tube (Weisman Lab) and run in the sonicator 3x for 20 seconds. (Invert tube in between each round)
Centrifuge at 4°C for 10 minutes.
Add 1.5mL of 1x HNG to GST and GST-EEA1 proteins. (-80°C)
Combine 450µL of protein with 450µL of lysates.
Place tubes end over end for 30 minutes at 4°C.
Add 50µL of glutathione sepharose beads to each tube.