User:Brigette D. Black/Notebook/Brigettes Notebook/2009/06/05/Making Antifade From Stock
CAT
- Stock concentration: 800 microgram/ml of catalase
I weighed out 9.9 mg of catalase and then added 12.4 ml of BRB80 (chilled on ice). There was some clumping in the mixture, so (although not the best thing to do for enzymes) I vortexed the solution until it was fully mixed. I attempted to filter the solution through a 0.2 micron filter, but it would not go through. Koch poured this into aliquots. These aliquots were then put in the -20C freezer.
GOD
- Stock Concentration: 2 mg/ ml of glucose oxidase
I weighed out 26 mg of glucose oxidase then added 13 ml of BRB80 (chilled on ice). In this case, there were some long thread-like things in the solution; I vortexed the solution, but it did not get rid of them, so I put the solution through a 0.2 micron filter. It went through easily and came out clear. I then poured it into aliquots which were put in the -20C freezer.
It is entirely possible that I made a little too much of both the CAT and GOD. 13 ml of each equates to 1300 "doses" (25 microliters) of antifade solution.
Antifade
From the new CAT and GOD components, I mixed up a new batch of antifade cocktail.
- 300 microliters CAT
- 300 microliters GOD
- 150 microliters BME
This made a total of 750 microliters. I then aliquoted this mixture into 30 aliquots each containing 25 microliters of antifade. Now, when we want to use antifade solution, there is already a 25 microliter cocktail readily available.
Using the microtubules the Lihn and Igor had polymerized this afternoon, I tested the antifade solution with good results. The microtublues were visible and bright enough to observe for about 90 seconds, which is actually longer than what I had observed on or previous occasions ( May 26).
