User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/23

ADA preparation

• All ADA procedures for this lab period was conducted by Puja Mody. The same approach was conducted during 09/19/2012
• This was a two day procedure continued by Michael Nagle on the following day, 10/23/2012
• The main concept for the procedure: E. coli was transformed to express ADA. After a 4 h. protein expression on an incubator shaker, the solution were centrifuge. The supernatant was collected and stored since this contained the soluble ADA while the pellets (cellular debris) were discarded.

Au/lysozyme solutions

• The calculations were supposed to be posted on the notebook of Michael Nagle.
• The calculation the author had executed for stock solution preparation is on 10/17/2012. The lysozyme stock solution was 15.24 μM and the gold was at 8.24 mM.
• It was decided that the lysozyme would be kept at a 1.5 μM concentration for all sample varying the Au concentration. The same mole ratios from Au/BSA were used 60, 80, 100, 120, 128, 130, 132, 133, 134, 136, 138, 140, 160, and 170.
• When the Au/lysozyme solutions were made, Nagle placed the rack in the Thermo Scientific incubator at 50°C for 4h.

AAS of Au/BSA solution of 10/17/12

• The Au/BSA solutions were taken out from the oven. Mole ratios 60, 80, and 120 were the only solutions that exhibited a consistent, clear purple color with no fibers. This was indicative that all gold nanoparticles were in solution and none have nucleated to BSA.
• Mole ratios 100 and others produced fibers, having solutions clear and colorless. The liquid was taken from each solution and transferred to falcon tubes; leaving some liquid suspending the fibers in the original tube. As a result, they were irregular amounts on each tube.
• The irregular amounts created difficulty for centrifugation. Hours was spent in balancing the liquid for each pair of tubes.
• Time went by and the fibers formed pellets on the bottom of the tubes. The formation of pellets was acceptable to allow to take AAS of the solutions.
• The program for the Shimadzu AA-6200 Atomic Absorption Flame Emission was set on autozero. The flame was ignited exhibiting a blue-green color.
• The blank used for the experiment was water.
• The standards were run followed by an interval of sample-blank measurements. The calibration curve can be seen on the data section of Puja Mody's notebook.