So it's your turn to load the FPLC?
Welcome to the Guide!
Purification
Step 1: Load the Superloop
Important things to consider. If you don't remove the caps then the plunger will not move.
To load the superloop you first want to flush out any extra lysate with water. With the syringe pull up more than 50ml of DI water, or water store water. Then remove the top cap of the super loop on the opposite end in which you want to flush out with water. On the side with extra lysate or where the plunger is closest to, unscrew the cap and screw on the syringe adaptor, then screw on the syringe, point the superloop down towards the sink, and inject with considerable force into the superloop. Once you are done flushing out with water recap both ends.
Take up more than 50ml of resuspension buffer in the syringe. Make sure you remove any bubbles from the syringe by holding it vertically and pushing out any air. It is normal for some of the buffer to spill out. Then unscrew the top cap and screw the syringe on the side where the plunger is closest and inject the buffer. It is important that all bubbles are minimized to reduce error on the FPLC.
Lastly, we need to load the lysate. Use the tube applicator to take up all the lysate in the syringe, you may or may not have 50ml. Remove any bubbles from the syringe try not to accidentially lose any lysate to this process. Then inject the lysate onto the side where the plunger is closest. Remember to remove the top cap!!!! After loading the lysate put both caps on the superloop then quickly move to the FPLC in the cold room.
Step 2: Attatching the superloop to the FPLC
Some things to note about the FPLC before getting into starting a run. If the lines have any kinks (sharp bends), the buffer will not be able to move through the tubes smoothly. The valve to load and inject is manual so you have to make sure you are there in person to flip the switch.
Start by placing the superloop in the clamp to hold it in place. Remove the top cap and screw on the first tube. Then remove the second cap and screw on the bottom line. Check to make sure that the lines have no kinks. The remove the bottom cap of the Nickel column and screw on the bottom line with the long attachment. Then unscrew the top cap and screw on the line. All the screws should be finger tight and to check that the lines are secure lightly tug on each end.
Step 3: Replace any microtubes
Check if there are any microtubes with any liquid. If there are discard in the beaker they will be cleaned and recycled. Replace any discarded microtubes.
Check the arm is in the correct position to collect fractions
Step 4: Buffers
Place line A into the Resuspension buffer. Line B into the Elution buffer. When removing the lines from the buffers rinse with DI water before replacing into the Water. It is important that the lines never run dry.
Step 5: Last checks before running the program
Check to make sure that the switch is on load before starting the program, the lines are in the right buffers, there are no kinks in the lines, there are no microtubes filled with water, and the arm over the microtubes is in the correct place
Step 6: Start the program
Turn on the computer, the password is chemlab.
Then click on unicorn...
From there connect the machine then click method run.
Select DesD Purification method.
Imput the volume of lysate in the superloop then click start.
Stick around for 5 min and wait for the computer to give the "manually flip" message
At this point flip the switch to inject and click confirm and continue. Then set a timer for 5 min less than your loaded volume that way you are ready to switch the FPLC back to load.
Once the FPLC give the same "manually flip" message flip back to load. After this the FPLC will start the wash and collect the protein. YAY
Desalt
So you got protein! Now you have to remove all the salt from your protein.
Step 1: Load the Superloop
Checking the concentration on the nano drop pick the highest 6-7 concentrated fractions of Ni pure.
Combine all the fractions in a falcon tube. Make sure to keep everything on ice.
Following the same protocol as before wash the super loop with 50ml of water pushing the plunger toward the 50ml line then backwash with you desalting buffer, pushing the plunger back toward the 0ml line. Once the plunger is back on the 0 ml line, inject your combined fraction on the 0ml side, you should have around 12ml of Ni pure since the column can only hold 12.5ml. Make sure you re cap the superloop before bringing it to the FPLC
Step 2: Attaching the Superloop to the FPLC
Same as before clamp the superloop to keep it in place screw on the top and bottom line and check to make sure the line is secure with a gentle tug test.
Step 3: Change out the microtubes
Change out any used microtubes discarding the used ones in the beaker for recyling. Replace the discarded microtubes with clean tubes from the boxes. Check the arm is in position to collect the fractions.
Step 4: Buffers
Place line A into the Desalt buffer. Line B into the milliq water. When removing the lines from the buffers rinse with DI water before replacing into the Water or changing buffer. It is important that the lines never run dry.
Step 5: Last checks before running the program
Check to make sure that the switch is on load before starting the program, the lines are in the right buffers, there are no kinks in the lines, there are no microtubes filled with water, and the arm over the microtubes is in the correct place
Step 6: Start the program
Turn on the computer, the password is chemlab.
Then click on unicorn...
From there connect the machine then click method run.
Select Desalt Hi-prep large column method.
Imput the volume of lysate in the superloop then click start.
Stick around for 5 min and wait for the computer to give the "manually flip" message
At this point flip the switch to inject and click confirm and continue. Then set a timer for 5 min less than your loaded volume that way you are ready to switch the FPLC back to load.
Once the FPLC give the same "manually flip" message flip back to load. After this the FPLC will start the wash and collect the protein. YAY
FPLC closing tasks
BEFORE YOU LEAVE
Please do these task to ensure the longevity of our FPLC
Place both of the lines in water.
Start a manual run with a flow rate of .5 ml/min, make sure all the lines are being flushed out by checking the graphic at the bottom left corner of the screen. The green line shows the flow through switch it to flow through the entire instrument.
Leave that running for 10-15min. Once the time is up disconnect the FPLC on the computer and close everything out.
You're all set have a good day!