User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/04/01/Osmotic stress planning
The first chemical I am going to use as an osmotic stessor will be betaine. I want to complete 3 initial points to the data taking. 2 extreme points and one point between. I already have my 0 mM betaine point so I will take data for the following:
- 1000 mM betaine
- 5 mM betaine
- 60 mM betaine
I understand that the 60 mM point isn't really between 1000 and 5 but, I am going to approach 1000 mM logarithmically. So if I approximately double the concentration from 5 to 1000, 60 is the middle point for me and what I plan to do.
Since I've adopted the term software to mean chemicals, biological materials, and solutions, I need to prepare a 2M solution of betaine in PEM. There are some things to take into consideration when preparing this solution.
- I don't want the betaine to directly interact with my casein. This means that no betaine will go into my casein stocks.
- Due to my preliminary data on caseins, I will use alpha casein for my surface passivator.
- Betaine will only be added to motility solutions. This means I only need 2M betaine in PEM. I will dub this solution PEM-Bine. (Ultimately I will look at sarcosine and proline and those solutions will be called PEM-Sine and PEM-Pine respectively.)
- Betaine is soluble to about 5 M in water. So, I'm going to make a 5 M stock solution of Betaine in PEM (PEM-Bine).
So I ran the 1000mM betaine experiment today. Below are two movies. Both are of gliding motility assays with alpha casein as the surface passivation. The second movies is with 1000mM betaine in solution.
I was really hoping for a dramatic affect but it doesn't look like there is one. Hmm...
- Steve Koch 23:53, 1 April 2010 (EDT): I re-skimmed the two osmotic stress-related actin/myosin papers. The paper which appears most thorough claims that the speed effects in gliding assays are due to viscosity changes, not osmotic stress. Maybe that's true for non-processive myosin? If it's true for kinsein (I'd be surprised) then using betaine (which has lower viscosity than sucrose) would not have much effect. I still think, though, that dramatically changing the off-rate of the motor domain will slow it down.
- Here's the paper that examines many osmolytes thoroughly with the gliding assay for actin: Viscosity and solute dependence of F-actin translocation by rabbit skeletal heavy meromyosin.
- Here's the paper that says osmotic stress affects actomyosin, but does not use gliding assay: Osmotic pressure probe of actin-myosin hydration changes during ATP hydrolysis.
- Steve Koch 23:59, 1 April 2010 (EDT): This paper looks very important, and I've not read it yet. Every Record paper I've read is very high quality, extremely detailed, and a great resource. Usually very complicated, though. Vapor pressure osmometry studies of osmolyte-protein interactions: implications for the action of osmoprotectants in vivo and for the interpretation of "osmotic stress" experiments in vitro. Here's a quote from the abstract: "Consequently, of the solutes investigated here, only betaine might be suitable for use in osmotic stress experiments in vitro as a direct probe to quantify changes in hydration of protein surface in biopolymer processes."