Purification of GST Fusion Proteins by GSTrap: Difference between revisions
Added initial protocol, need to add details about HPLC Run |
→FPLC Run: Added details about HPLC run |
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==FPLC Run== | ==FPLC Run== | ||
#Fill | #Fill tray with ice, or put PBS and Glutathione in ice bucket. | ||
#Start the pump, by going to | #Turn computer on and login if necessary, username=saltielhplc; password=eatAdEv3 | ||
#Open TC Navigator and login | |||
#Go to Apps -> Kickoff -> GST Purification | |||
#Enter number of samples you want to run | |||
#Enter sample names and click through to finish | |||
#Start the pump, by going to Hands On, Start Pump | |||
#Turn on lamp if necessary by going to Detector -> Turn Lamp On within the Hands On Window | |||
#Rinse the leads with water and place in PBS (A) and Glutathione (B) | #Rinse the leads with water and place in PBS (A) and Glutathione (B) | ||
#Prime the leads by first selecting 100% A on the Hands On Window and while the pump is running, loosen the black screw in the lower section (remove the panel). Attach a 50 mL syringe and pull through 5-10mL | |||
#Switch the pump to 100% B (Glutathione solution) and prime those lines. Switch back to 100% A (PBS) | |||
#Connect the GSTrap column and let equillibrate for a few minutes | |||
#WIth the injection loop set to load, using a syringe inject ~5mL of lysate into the 5mL sample loop | |||
#Turn the loop clockwise to inject to start the run | |||
#After 30min, the fraction collecter will automatically start, make sure it has 2mL tubes without caps ready | |||
==Post-FPLC== | ==Post-FPLC== | ||