User:Brigette D. Black/Notebook/Brigettes Notebook/2009/06/15/Ascorbic Acid With Rhodamine Tubulin
With the TRITC made, we decided to try to look at it plain, with antifade, with ascorbic acid, and with ascorbic acid and antifade. There are videos of the latter three on the server.
Plain (without ascorbic acid or antifade)
I polymerized the TRITC by placing it in the thermal cycler at 37 C for 20 minutes. We then stabilized it with 99 uL of BRB80-T.
Under the microscope the microtubules seemed fairly healthy, the appeared perhaps slightly longer than the FTIC MTs. I was able to observe them for about 1 minute with the room lights off before they had photobleached; in that time I did not see any breaking.
- 85 uL BRB80-T
- 2.5 uL AF (make 6/5)
- 2 uL 1.6M dextrose
- 10 uL stabilized microtubules
These appeared to be pretty healthy too. The antifade kept them from photobleaching for about 6 minutes (observable without room lights). I believe for the camera they were visible for about 3 minutes. I did not see any breaking, nor were we able to record any of the microtubules breaking.
With Ascorbic Acid
- 50 uL stabilized microtubules
- 1 uL 0.27 M ascorbic acid
This mixture gives about a 6mM concentration of ascorbic acid (I was aiming for between 4mM and 8mM). Under the microscope this solution looked a little different than just the plain TRITC, the microtubules appeared to be slightly shorter and the background was slightly cloudier with multiple bright dots. I am assuming that these were the remnants of mictotubules that had broken before I could get a good look at them. The sample photobleached in about a minute.
With Ascorbic Acid + Antifade
I pipetted out 50 uL of the Antifade + microtubule concoction (listed above) and then added 1uL of 0.27 mM ascorbic acid, for a total of 6mM ascorbic acid.
This was probably the most disappointing of the four combinations. There were relatively few microtubules in the field of view (I think 6 in the field of the camera), and the background was once again cloudy and littered with bright dots. The sample photobleached in about 6 minutes, consistent with previous observation of the sample with just antifade.
It occurs to be now that this could be undissolved bits of the vitamin C powder, however I vortexed the ascorbic acid multiple times, the solution appears clear , and the concentration is significantly lower than the solubility of vitamin C in water. Another possibility is that the pH of the solution is causing the microtubules to fall apart very quickly, perhaps suspending the vitamin C in BRB80 instead of DI water would fix the problem.