GST Pulldown Assay: Difference between revisions
wrote initial protocol |
added note about pulldown time |
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#Take out a 50 uL aliquot of each lysate sample and add 50 uL of 2x sample buffer as a lysate control. | #Take out a 50 uL aliquot of each lysate sample and add 50 uL of 2x sample buffer as a lysate control. | ||
#Add the appropriate amount of lysate to each set of beads | #Add the appropriate amount of lysate to each set of beads | ||
#Place tubes end over end for 30 min-4h at 4°C. | #Place tubes end over end for 30 min-4h at 4°C. Use 30 min for time sensitive interactions (ie GTPase-effector) and longer time for more stable interactions. | ||
#Add 50µL of glutathione sepharose beads to each tube to increase the bed volume of beads and to limit accidental aspiration of beads | #Add 50µL of glutathione sepharose beads to each tube to increase the bed volume of beads and to limit accidental aspiration of beads | ||
#Wash each tube with 1x HNG five times at 4°C. | #Wash each tube with 1x HNG five times at 4°C. | ||