GST-EEA1 Pulldown from Yeast

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

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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.