View source for 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.