User:Brigette D. Black/Notebook/Brigettes Notebook/2009/06/11/Microtubules + Ascorbic Acid

Why use Ascorbic Acid
In the paper Mechanism and Dynamics of Breakage of Fluorescent Microtubule by Gao et al there is a claim made that the addition of ascorbic acid can prevent MTs from breaking apart. They suggest that free radicals can cause MTs to break, and since ascorbic acid removes free radicals, adding a small amount to MTs is a good way to test to see if free radicals exist within a sample and if they do, lengthen the life of MTs.

They used two concentrations of ascorbic acid, 4mM (mmol/L) and 8mM. They claim that the 4mM concentration The 8mM solution extended the life by at least 6 minutes (the MTs had been photobleached by such an extent that they were no longer observable). I decided to try adding ascorbic acid to our microtubules to see if there was a noticeable improvement in the lifetime of our MTs.

I made a .273 M solution of ascorbic acid using powered vitamin C (144 mg) and DI water (3 mL). I made a high concentration so that when it is added to the MTs the density of MTs remains nearly the same.

Without Antifade
I divided the aliquot of MTs and BRB80-T into two aliquots, each 100uL in volume. I then added 1.5 uL of the ascorbic acid to one (to make a 8mM solution), and then 1uL to the other (this is actually about 6mM, not 4mM, but I thought that there should still be a noticeable difference).

I tried to look at the two concentrations under the microscope, however they both photobleached so rapidly that I could not witness any MTs breaking. I did notice that if the room lights were turned off that the MTs could be observed for much longer (probably about 3 minutes). I did not however get to witness any breaking with either concentration, however.

With Antifade
In an attempt to conserve resources, I used the 8mM ascorbic acid MTs and mixed this in with the antifade cocktail. Lihn mixed up the antifade MT solution using the antifade stock created last week. There were about 7.2 *10^-6 g of ascorbic acid in the solutions, but in order to make it a 8mM solution, there needed to be 1.4*10^-4. So I added about 2 uL of the ascorbic acid (9.6*10^-5 g). The final solution was around 6mM of ascorbic acid. I was a little sloppy with adding the ascorbic acid and doing back of the envelope type calculations to get the concentration within range, but not really aiming for 4 mM or 8mM specifically.

When I looked at this under the microscope, however, I saw nothing! There were no MTs anywhere. However, some photobleaching could be observed as the uniform field of view would turn slightly darker over time. I thought perhaps that there were not enough MTs, so I began adding more to the antifade solution (about 2uL more at a time). However, even after adding a total of 10uL more of the MTs, there were none to be seen.

I do not know exactly why this happened. The best guess I have is that the ascorbic acid reacts in some way to the enzymes in antifade solution that results in instant MT death. I mentioned this to Andy who said he would think about it and hopefully we can come up with an answer. I will perhaps try do this again as perhaps something went wrong in the mixing of the antifade and MTs.