enter protocol here== '''Materials''' ==*2x HNG (100mL). Combine 100mM HEPES (10mL), 300mM NaCl (7.5mL), 20% glycerol (20mL) *1x HNG (10mL). Mix 5mL 2xHNG and 1 PI tablet. *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. *Wash each tube with 1x HNG five times at 4°C.*Load in a 4-12% gel.
