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GST-EEA1 Pulldown from Yeast

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Contents

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.
Last edited 13 years ago by Sheelak