4
edits
Changes
added categories and link to GST-GTPase pull downs
[[ Category: Protein Purification ]]
[[ Category: Protein-Protein Interactions ]]
[[ Category: Protein Biochemistry ]]
[[ Category: Proteins ]]
==Materials==
*2x HNG (100mL). Combine 100mM HEPES (10mL), 300mM NaCl (7.5mL), 20% glycerol (20mL)
#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
#Place tubes end over end for 30 min-4h at 4°C. Use 30 min for time sensitive interactions (ie GTPase[[GST-effectorGTPase Pull Down Assay]]) 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
#Wash each tube with 1x HNG five times at 4°C.