Preparing an LCP Mix

In order to make an LCP mixture you will need the following equipment:

Two 100 µL gastight glass syringes with a coupler

  1. A 10 µL gastight glass syringe with dispensing needle
  2. Syringe repeating dispenser
  3. Heating pad
  4. Monoolein
  5. Protein sample
  6. 100% wash ethanol (EtOH) in 50 ml centrifuge tube
  7. 5 cent piece
  8. Flat bladed tweezers/regular tweezers
  9. Acupuncture needle for cleaning

1. The cubic phase is obtained when the weight ratio of aqueous protein sample to monoolein is 40:60. We assume that both the protein sample and the monoolein have densities of 1 mg/µL and we use a 40:60 volume ratio to obtain the cubic phase. (The density of monoolein is reported to be 0.942 g/ml, and the density a protein solution will vary). All steps must be done at room temperature, as the cubic phase is temperature dependant.

2. Ensure that the three syringes are clean and dry. If not, draw up 100% EtOH into syringes with couplers attached and push EtOH through the couples repeating several times. Blow air through the syringe to dry out the barrel. To ensure that connections are leak-free there needs to be a white Teflon gasket on the needles/couplers or in the syringes.

3. Check that there is monoolein in the heater block. This lipid should be stored in small aliquots, under nitrogen or argon at -20ºC. It melts at 37ºC. It can be melted and cooled a number of times without degradation.

4.  Warm one of the 100 µL syringes.

5. Prepare the small (10 µL) syringe/dispenser system – by attaching the PB-600 dispenser to the syringe. Remember to shim the plunger, and to make sure that the plunger and syringe are as co-axial as possible (that is, don’t bend the plunger)

6. Load the warmed 100 µL syringe with melted monoolein by pipetting 27 µL of the lipid into syringe from the threaded end. Draw down the plunger as you pipette in the lipid. Ensure that there are no air bubbles caught in the melted lipid, and remove any that may have been trapped by pushing the plunger rapidly. Use your fingers as a stop to prevent the lipid from shooting everywhere. Very gently push the plunger so that the lipid is all the way at the end of the syringe. Look down into the syringe to see the change in meniscus as the lipid reaches the end of the syringe. Attach the coupler (with Teflon spacer) to the end of the syringe, and push the plunger gently until you see lipid just start to emerge through the coupler. The coupler contains 1 uL – make a record of how much lipid you have loaded. Keep the loaded syringe/coupler warm.

7. Load the other 100 µL syringe with 16 µL (or the appropriate amount, given the volume of the lipid you loaded) of protein solution. Load into the syringe from the threaded end. Draw down the plunger as you pipette in the protein. Remove any bubbles in the protein solution in the same way that you removed bubbles from the lipid. Gently push the protein to the end of the syringe – check this by looking down the barrel of the syringe as you adjust the plunger, you should see the change in meniscus as the protein solution gets to the end of the syringe.

8. Attach the lipid-containing syringe with the coupler to the protein syringe. Ensure that every joint has a white Teflon gasket in it. Make all the connections finger tight, but please don’t strip the threads on the coupler or the syringes.

9. Mix by pushing carefully on the plunger from the lipid side so that all the lipid solution enters the protein-containing syringe. Then push the protein/lipid back into the other syringe. Continue this until the sample has been pushed through the coupler ~100 times. Hold the two syringes horizontally and push one plunger at a time. It is easy to burst the syringes doing this – as the mixture is very viscous. This is why we pre-warm the syringe for the monoolein, and start mixing ASAP after the monoolein has been loaded.

10. The mixture may take some time (an hour or so) to form the cubic phase. The transition can be monitored, as the mixture turns from being opaque to translucent. Wait until this transition has occurred before dispensing the mixture.

11. Once the mixture is homogeneous, push all the mixture into one syringe. If you are preparing the sample for us to use in a LCP experiment you can bring in the syringes at this point.

12. Ensure that the plunger can move in the barrel of the 10 µL syringe by moving it up and down, leaving the plunger pushed all the way in. Disconnect the empty 100 µL syringe from the coupler, attach the 10 µL syringe to the adapter/coupler and slowly push the mesophase into the small syringe***. As you load the mesophase into the dispensing syringe the plunger on the syringe will extend out. (Loading may require a bit of force, but a gentle, sustained force – this is another fabulous opportunity for bursting syringes). When you have loaded the small syringe (no more than 10 µL), disconnect the coupler, and replace with the dispensing needle, checking that there is a Teflon spacer in the right place. Move the plunger assembly to just under the tab of the extended plunger. Tighten down the screw of the plunger assembly. You should now have your 10 µL syringe full of lipid/protein mixture and connected correctly to the dispenser as seen below. Click on the dispenser until you see LCP emerge from the end of the needle. (Count the number of clicks)

13. Rejoin the two larger syringes through the coupler. This will keep any remaining LCP hydrated in case you want to re-load the smaller syringe with mesophase