User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/07/26/Whole casein failure

W-PEM
So, I figured out that there is a shelf life associated with the solutions that I prepare for my gliding motility experiments. It turns out that there is about a 6 month shelf life for all solutions containing PEM in them. This necessitated that I prepare all new solutions. Those new solutions included: Of course, the PEM solution containing whole casein (W-PEM) is the most difficult to prepare. The difficulty is compounded even more when you try to make W-PEM with the wrong type of casein as I will explain more below.
 * PEM
 * alpha-PEM
 * beta-PEM
 * kappa-PEM
 * PEM-Glu
 * PEM-A
 * W-PEM

This type of whole casein from bovine milk has "essentially no vitamins" (Sigma's wording) in it. It just does not, under any circumstance, go into a solution of PEM. I've stirred and heated (80-95C) the solution for approximately 5 hours to no avail. Of course this was unusual to me since I was able to get whole casein into a solution of PEM 6 months ago with relative ease. So, I didn't understand what was going on with my prep this time and guess what, I didn't write it down when I prepared the sauce 6 months ago.

The first failed attempt I tried to reproduced what I did 6 months ago. I stirred 0.5 mg/mL casein in a beaker of PEM and heated it to around 60C. The result...nothing went into solution and my liquid evaporated. So, the second time I did it, I got wise and ordered a condenser. This would allow me to let the solution boil for basically forever without having to monitor the evaporation of my PEM solution.



You can see the temperature I have the solution at. The PEM + casein is in the flask with a stir bar in it. Connected to the flask is a manifold (the tall thin glass thingy) that keeps the contents of the flask open to the atmosphere. Hence the reason I'm able to make temperature readings. I then pump cold water through the manifold using a fish tank pump. What happens is that the solution in the flask may evaporate but, when it comes in contact with the cold manifold, it condenses back into solution. I've used this setup before quite successfully for ion exchanges in Sephadex columns that require boiling the solution for over 6 hours at a time.

So, I used this setup for the vitamin free casein after my first failed attempt. I then let it go for about 1 hour and the casein didn't go into solution. I swore to myself that last time it only took half an hour to get everything into solution so I did the only reasonable thing I could think of, I increased the temperature. Nope. Still didn't work.

After receiving an email from Koch about how I have been slacking on my notebook entries, I started a thread in Friendfeed talking about what is and what isn't appropriate to add to your notebook. Dr. Neylon made a spectacular comment about how writing more never hurts when it comes to your notebook. He referenced an example about preparations of buffers in his lab that were not documented efficiently and thus they are now having problems with them. How fortuitous for me it was to make this thread since this is exactly what is happening to me. I didn't write in my notebook 6 months ago how I made the whole casein PEM solution and now I can't remember exactly how to do it. Hence my inability to get whole casein into solution.

Bill Hooker also commented on how every engineering nightmare one has to go through in the lab should be documented in your notebook. No matter how silly you might think it is. Well Bill, I am now on the band wagon for updating my notebook with just about everything I do from now on because the solution to my buffer problem is so stupid simple that I could have saved myself 3 days worth of irritation if I just wrote the bloody thing down in the first place.

I'm using the wrong casein!

The correct casein to use is this one. This casein I guess has vitamins in it. I have absolutely no clue as to why this casein goes into solution and the one above does not. That's a mystery I'm not willing to solve right now. I started stirring 0.5mg/mL of this casein in PEM at room temperature and brought the temperature of the solution up to 60C of which then I turned off the hot plate. The max temp the solution came to was 63.5C. At the 15 minute mark (which was right about when the solution reached 60C), I stopped the stirring and looked at my solution. Guess what. All the casein went into solution.

Needless to say, I'm infuriated at myself for not writing this down earlier. And yes, the old adage stands; 2 weeks in the lab will save you 2 hours in the library. If I only wrote my procedure down the first time, I would have been able to search for it and not have wasted 3 days of my time trying to get the wrong casein into solution.