Comparative Physiology @ Guelph
Column Run for Pacific Hagfish Thread Proteins
- Lyophilized thread protein stored in freezer in a container with desiccant
- AKTA FPLC with fraction collector
- Column packed with Q Sepharose Fast Flow and evaluated to ensure proper packing
- BD 10 ml Syringe with Luer-Lok Tip
Note: All buffers and solutions ran through the FPLC must be filtered and degassed using a maximum of 0.45 ul filter paper and a filtering apparatus. This ensures that the system does not become clogged with particulates, and that no gas bubbles interfere with the column run.
- 20% ethanol
- 1M NaOH
- 2M NaCl
- 8M Urea solution (20 mM Tris Base HCl, 1mM EDTA, 8M Urea, 1mM DTT)
- pH chosen b/w 8.0 and 8.5, make about 1.5 L, must be made fresh
Note: Solublize Tris and EDTA in about half the amount of final volume desired, then add urea –this will bring the solution close to the desired volume. Adjust to the pH desired using HCl. Filter. Add DTT at the latest possible time before use.
- 8M Urea solution with KCl
- Before filtering the Urea described above, remove 0.5L and add KCl to make the solution b/w 100mM and 200mM depending on the salt concentration desired. A lower salt concentration gives more precise control over the gradient, but takes longer to run and may not elute out high amounts of protein. Filter once KCl is completely solublized, and add 1mM DTT before use.
- First thing in the morning, begin preparing the Urea. It must be made fresh to avoid degradation, and cannot be heated to aid in solublization. It will take approximately 2.5 hours to solublize and filter.
- Turn off the fridge if it is on. This run must be done at room temperature to keep urea from crystallizing.Remove lyophilized protein from the freezer and allow it to come to room temperature in an airtight container with some dessicant to absorb any water particles.
- Prepare the FPLC
- Turn on the computer and the FPLC. Use Todd’s profile. Open the program Unicorn from the desktop. Username: default, Password: default. Ensure the lamp is turned on to warm up.
- Prepare the Superloop
- Ensure the lines A and B are in ethanol.
- The column should not be attached while cleaning, instead use the brown wire connection that connects port 1 with the sensor.
- From the system control screen, choose Manual, Flowpath, choose load from the dropdown menu and click Execute twice. Load 10 ml of 20% ethanol into the superloop using a syringe. Avoid forcing air bubbles into the line by removing any air from the syringe, and making a drop-to-drop connection. Watch the piston in the superloop rise.
- Choose Inject from the drop down menu, and click Execute twice.
- From the manual controls on the FPLC, set the flow rate to about 5 ml/min, and the %B to 50%.
- Watch the piston in the superloop carefully, and be prepared to pause the machine just before the piston reaches the bottom of the superloop.
- Repeat steps iii to iv twice more with 20% ethanol, then 3x with 8M Urea once it has been filtered.
- Prepare the Column
- Attach the column to the setup by first removing the brown tubing and end pieces. Insert small black end pieces to the Inlet 1 and the sensor. Remove the column from cold storage and place it in the holder in position so that if it drips, it will not affect the sensor box. Remove the cap from the top of the column, and run the pumps at 1 ml/min so that the black end piece fills with eluent. Once it is screwed in tight, attach the bottom endpiece.
- If unsure if it has already been done, clean the column with 3 column volumes of 1M NaOH. Do this from the manual controls choosing 5ml/min, 50 % B and setting the timer to end after the calculated amount of time necessary for 3 column volumes. Place feed lines A and B in the NaOH solution and select run.
- Recharge the column by running 3 column volumes of 2M NaCl using the same settings as for the NaOH. Be sure to allow as much NaOH as possible run off by resting the tips of the filters against the lip of the bottle when changing between eluents.
- Run 2 column volumes of degassed water through the column in the same way.
- Prepare Fraction Collector
- Ensure fraction collector has fresh new 5 ml tubes where the desired fractions are expected. Washed tubes are acceptable where no peaks are expected.
- Place tubes in the collector set up, and ensure the proper tube size is selected, the tubes come up to the line on the back of the swing arm, and that the tubes are lined up so that the drip lands in the centre of the tube.
Note: Manual fraction collection can also be used if the fraction collector is being unreliable. Collect peaks from the orange tubing before it reaches the fraction collector into falcon tubes.
- Prepare Protein
- Once urea is prepared, and the protein has come to room temperature, measure 0.030 g of protein and dissolve it in 12 ml of 8 M urea. This may take approximately one hour. Once dissolved, heat to 60 degrees Celsius for 5 minutes.
- From the system control screen, choose Manual, Flowpath, choose load from the dropdown menu and clickExecute twice. Load 10 ml protein into the superloop using a syringe. Avoid forcing air bubbles into the line by removing any air from the syringe, and making a drop-to-drop connection. Watch the piston in the superloop rise.
- Ensure Program is Ready
- Be sure the correct program is ready to go if an automatic run is to be used. If a manual run is to be performed, ensure the plan is well thought out.
Note: The most precise run completed to date was Manual run 6. It can be used as a starting point for planning a new run. A good run to start from if planning an automated run is MM PHF 4B001.
- Prepare Eluents
- Place line A in 8M urea, and line B in 8M urea with KCl. Be sure to allow as much NaCl as possible run off by resting the tips of the filters against the lip of the bottle when changing between eluents.
- Start the Run
- If doing a Pre-Programmed Run – On the system control page, choose Run, then select the method that was prepared. Follow the screens, checking the column volume specified, the fraction size, the wash-through volume etc… and answering the questions.
- If Doing a Manual Run
- Autozero the lamp.
- Equilibrate the column with 2 Column volumes of 100% A.
- Inject the sample from the superloop. Run 2 column volumes of A to wash through any unbound protein.
- Without pausing the FPLC, change the concentration of %B as desired. Be sure to run a high concentration of salt at the end of the run to remove as much protein as possible.
- Monitor the Progress
- Keep an eye on the FPLC while running, periodically checking that the max pressure is not exceeded (be concerned if the pressure reaches 0.5, consider changing the filters), that the waste is not overflowing, and that the fractions are being collected accurately.
- Once the run is complete label the fractions appropriately. Add methylamine HCl to the tubes to make the solution approximately 10 mM. Store the fractions in the fridge immediately, or freeze portions of the samples of interest in 2 mL capped tubes if the samples are not to be used within the next day or two. The FPLC run can be printed out, as well as a summary of the parameters specified.
- Clean out the system of all urea. If the urea is not rinsed completely, it crusts and causes problems in the machine. Be sure to wash out all bound protein by running 1.5 column volumes of high salt concentration, then washing with 1.5 column volumes of NaOH. Finally rinse the column with 2 column volumes of 20% ethanol. Alternatively, a preprogrammed cleaning run can be used. Remove the column and store in the refrigerator. Connect the brown tubing to bypass the column. Rinse the connection to the fraction collector, and finally, spray down any urea that may have dripped onto the machine.