User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/08/19/Surface passivation experiment

Goals

 * Polymerize new microtubules.
 * Need to make a new stock of BRB80T.
 * Check to see if the kinesin is still active.
 * If not, get a new batch.
 * Make a new stock of whole casein.

Experiment
Observe the gliding motility assay for:
 * 1) $$\kappa$$-casein
 * 2) whole casein
 * 3) spin coated lipid substrate
 * 4) silicone substrate
 * 5) lipid coated silicone substrate

Whole casein
So none of us has been able to get whole casein to go into solution. Casein exhibits amphiphilic behavior and will make micelles once it is in an aqueous environment. To try and make it go into solution, I will try putting it into a basic solution and if it goes in, I will then prepare the solution so that it has the components of BRB80 in it. Of course this will mean that the casein solution will have excess Na2+ ions in it and correspondingly more Cl- ions when I pH it but, I will just have to check to see if it is okay or not.

First try: The first thing I am going to do before trying to change the pH is to try and heat up the BRB80 I have. Going with what Wikipedia says about casein not denaturing, I am going to crank up the heat to around 90˚C to see if it will go into solution. The required concentration of casein needed is 0.5 mg/mL. This means I need 12 mg of casein. This is a pain to measure but it is my starting point.
 * I washed an 80 mL beaker and rinsed it with 5 volumes of nano-pure water.
 * I washed a thermocouple and a stir bar doing the same.
 * I have 25 mL of BRB80 in the beaker and it is stirring at level 5.
 * The heat is on and I'm sure it is going to take me forever to figure out how to set it such that it stays below boiling. Koch question: Can we get a new stirrer/hotplate that comes with PID temperature control?

Note: I totally forgot that the water is going to evaporate. Sometimes I amaze myself...

I was able to weigh out 15 mg and guess what, that's what I'm going to use. The temp right now is 60˚C and I'm going to sprinkle in the casein.
 * 11:30

Well I've got the temp at around 60˚C pretty stable. I also have the thing spinning at the fastest rate (10). It seems that everything is going into solution. I've sprinkled the casein in and now I'm letting it stir for a bit to see if I can get it all into solution.
 * 11:40

Yep. It all went into solution. I'm not very satisfied with the whole "casein won't denature" thing so in the future I think I will keep the temperature of BRB80 at body temp. But, if this works then I don't really care about the temp.
 * 11:45

I'm going to wait till it cools down to transfer it into a vial. I'll add any water back to solution that evaporated.
 * 11:55

I lost approximately 5 mL of H2O and added it back in. I will note that the solution formed some sort of bubble film on the top of the water. This is a clear indication that there is protein in solution. Whether or not that protein is denatured or not is yet to be determined.
 * 12:15

Update
I shouldn't have thought that the casein would not denature due to an increase in temperature. This thought was silly but, trying to get it into solution by increasing the temperature was not a bad idea, just a silly one.

This paper done in the 40s states that casein apparently doesn't denature when you heat it up. However, a simple Google search shows that denaturation does occur but I think the papers are talking about denaturing of whey proteins. Eh. I'm done looking into this and I'm just going to use it to see what happens.
 * 12:30