User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/04/24/More on kinesin & microtubule buffers
As we know, casein comes from milk. So, it would probably be a good idea for me to understand some basics about milk. I found a rather nice introductory website on utter puss and, it can be found here. I want to list some highlights from this page. Unfortunately the web page is copyrighted and I initially made some notes by copy pasting things from the site. I asked permission but Dr. Goff has not responded so I'm going to remove the quotes from the page and summarize my findings below. All information below can be found on Dr. Goff's site.
- Over 98% of milk fat is made up of triglycerides.
- The web page gives a breakdown of the fat content naming each constituent and rough percentages.
- Note: Milk is mostly saturated fats with oleic fat being the predominant unsaturated fat. See the Wikipedia article for a more complete description of fatty acids and their nomenclature.
- The fat globules in milk range in size from 100 nm - 15 µm. Homogenization causes these fat globules to decrease in size thus allowing things like casein to coat them to reduce their surface tension.
- I can see why you would want to do this for milk, but I do not understand why you would filter casein with ~ 200 nm filters. It just doesn't seem like a necessary step unless you want to make monodisperse micelles of casein.
- About 80% of proteins in milk are caseins. Since milk is about 98% fat, this leaves 2% of stuff available of which 80% of the 2% left over is casein. Not a lot.
- Note: It is difficult to precipitate the calcium out of the casein. If we use casein, we may have to use EGTA.
- This site confirms my previous statement that caseins are the vehicles that deliver calcium phosphates to young mammals.
- You can purify casein by electrophoresis.
My thoughts on this site
Good site with lots of good info. Sorry I couldn't put some of the more interesting numbers in my notebook. All of the above information can be found in Wikipedia but, not all of it is in a nice convenient spot.
I still cannot think for the life of me why we can't use a lipid bilayer to coat the glass in stead of casein. If all casein is doing is supporting kinesin and blocking the glass, then lipids will work. Plus, the added advantage to using lipids is that we know exactly what the lipids we use are. Then, we can add other things to the buffers that we want. Right now using casein means we will have an unknown quantity of calcium in solution and as we know, calcium and microtubules do not mix.
Andy Maloney 12:11, 25 April 2009 (EDT):
Dr. Goff did get back to me and he said I can use some of his info here! This is nice because he has some information nicely cataloged on the site I'd like to put here. So, more to come. Thanks Dr. Goff!
- Steve Koch 14:47, 25 April 2009 (EDT):Kudos to Dr. Goff! Above, you mention how the wikipedia article could be improved -- I say do it when you have time! I edit wikipedia all the time. It's the same software that OWW uses, so you should find it pretty easy. Also, about the lipid bilayer coating -- that sounds great. I have no experience in it. One thing that comes to mind is that full-length kinesin tails insert into lipid bilayers, correct? I.e. for vesicle transport. I would think there must be some background literature in this?
- Andy Maloney 18:49, 25 April 2009 (EDT): Well, from what the previous paper I read suggests is that kinesin does "bury" itself into a bilayer of casein. Whether or not it is necessary to make a monolayer of lipid first and then add kinesin, and then complete the bilayer, has not been answered. But, I will look into the kinesin inserting into lipid bilayers. Do you know what the tail looks like? There may be functional groups on it that like to be in nonpolar environments and this is why it sticks itself into lipid bilayers. Lipids are kind of expensive, but we can get some in vials that will basically last forever if not opened. I can see us doing things with lipids mainly because it seems that they act just like protein blockers for glass with the added benefit that they aren't proteins.