GST Pulldown Assay: Difference between revisions
added note about pulldown time |
added categories and link to GST-GTPase pull downs |
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[[ Category: Protein Purification ]] | |||
[[ Category: Protein-Protein Interactions ]] | |||
[[ Category: Protein Biochemistry ]] | |||
[[ Category: Proteins ]] | |||
==Materials== | ==Materials== | ||
*2x HNG (100mL). Combine 100mM HEPES (10mL), 300mM NaCl (7.5mL), 20% glycerol (20mL) | *2x HNG (100mL). Combine 100mM HEPES (10mL), 300mM NaCl (7.5mL), 20% glycerol (20mL) | ||
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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. Use 30 min for time sensitive interactions (ie GTPase | #Place tubes end over end for 30 min-4h at 4°C. Use 30 min for time sensitive interactions (ie [[GST-GTPase 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 | #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. | ||