User:Andy Maloney/Kinesin & Microtubule Page/W-PEM

Recipe

PEM
1.0mg/mL whole casein.
I will typically prepare a total volume of 25 mL. This means that I will weigh out 25 mg of whole casein to add to the 25 mL of PEM.

Procedure

When I prepare the whole casein solution, I use a condenser since I will be heating up the solution. The condenser allows me to heat up the solution without having to worry about evaporation. I've used the condenser for ion exchange before which requires you to boil things for several hours. These things work quite well as I was able to boil my column media without having to watch it the entire time in order to add water back to the solution.

Using the condenser is pretty straight forward. Here are the steps I take to set it up properly. Do not plug in the pump until the step to turn it on comes up!

1. I setup the condenser on a support beam with two chemistry clamps supporting it.
2. I place a stir bar in the ground glass neck flask and position it such that the magnet stirs evenly in the flask. If I were to boiling things for extended periods of time, I'd put some petroleum jelly on the ground glass part of the condenser. This helps to make a seal between the flask and the condenser and will prevent any condensation from the outside of the condenser from getting into my sauce as it boils. Thankfully, the W-PEM solution does not need to be on the heat for more than 15 minutes.
3. I pour in the casein to the flask making sure to not get any casein on the neck.
4. I then pour in the PEM to suspend the casein to a final concentration of 1 mg/mL.
5. I attach the condenser to the flask making sure that it is level and there is no strain on the glassware.
6. I attach hoses to both nipples on the condenser.
7. I then attach the bottom hose to the fish tank pump and submerge the pump in ice water and secure its power cord with a clamp. It's important to attach the bottom hose to the pump. That way the coldest water hits the condenser first.
8. This next step is important because if I run the pump without water flowing through it, I will burn it out. I use a syringe to suck out the air in the system and get water in through the condenser and out the top hose.
9. I now plug in the pump. The water will flow up the condenser and out the top hose. The cold water will cause the evaporation from the flask to condense back into solution. Sometimes there are a few air bubbles in the system initially. This is okay, I just tap the hoses to try and get out as many bubbles as possible.
10. I stir at a setting of 10 and heat on a setting of 3. I then let the system evolve for 15 minutes like this. The temperature in the flask will reach any where from 60 - 80°C depending on how much solution you are mixing up. I typically will make 25 mL total volume.
11. After 15 minutes, I shut off the heat and allow the solution to continue stirring until the hot plate has cooled completely.
12. Once the hot plate is cool, I take the flask off of the hot plate, put some Parafilm on the top of it and store the solution in the 4°C fridge overnight. This is to allow the protein that has bubbled up when stirring to settle back into solution.
13. The next day, I aliquot the W-PEM into convenient aliquots (without filtering) and store in the 4°C fridge for future use.