So it's your turn to load the FPLC?

Welcome to the Guide!

 

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. No tube can have any kinks, the buffer will not be able to move through the tubes smoothly if there are any kinks. The valve is manual so you have to make sure you are in person to flip the switch. 

Start by placing the superloop in the clamp and clamping down to hold it in place. Remove the top cap and screw on the first tube. Then remove the second cap and screw on the second on the bottom. Check to make sure that the lines have no kinks. The remove the bottom cap of the Nickel column and screw on the cap. 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. 

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 ...(the line over the microtubes)

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.