User:Monika Gasiorek/Notebook/CHEM-571 2014F/2015/03/31

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March 31st, 2015

New Reaction/Sampling Protocol

1. Prepare AuNP fibers - vortex each 5 mL test tube containing fibers and combine some necessary amount into a large falcon tube.

2. Centrifuge the falcon tube allowing the fibers to settle at the bottom and pour off the supernatant liquid.

3. Add Tris/CaCl2 buffer and vortex again, resulting in a homogenous solution of fibers which can be equipartitioned between 20 sampling epi-tubes.

4. Add protease to each epi tube. (1 epi-tube per each timepoint being sampled)

5. At 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60 min stop the reaction by removing from the "incubator/shaker" and centrifuging for 30 sec.

6. Collect the supernatant for analysis.

NOTES: Upon addition of the buffer in step 3, the fibers began to aggregate into clumps thereby undermining the desired homogeneity of the solution. The sonicator was used to try to break down the clumps, but the questionable state of the fibers (it appeared as though they might have been broken down into a colloidal mixture) after its use instigated a plot twist...

NEW PLAN: Vortex each 5 mL test tube of fibers (the solution seemed homogeneous at this point in time). Given a necessary volume of 0.053 mL of protease solution in a 1.0 mL sample total for a final protease concentration of 1 uM, 0.947 mL of the 1 mL sample volume consists of the fibers in 50 mM Tris-HCl/20 mM CaCl2 pH 8 buffer. So, take 0.927 mL of the homogeneous fiber sample from the vortexed 5 mL test tube, pour into an epitube with 0.020 mL of a 2.5 M Tris-HCl/0.5 M CaCl2 pH 8 buffer. Then add the 0.053 mL of protease for a total volume of 1 mL.

New Sample Analysis Protocol

1. Remake a Bradford calibration curve from 1 uM proteinase K samples with varying concentrations of lysozyme protein.

2. Develop a calibration curve (abs. v concentration) from which protein concentration could be determined corrected for the presence of protease.

3. Follow the same sampling Bradford protocal used before! (250 uL sample, 200 uL Bradford, 550 uL buffer)

Solution Prep

(1) 50 mL of 2.5 mM HAuCl4 solution was made from 0.04361 g of HAuCl4 (MW 339.785 g/mol) in 50 mL of distilled H2O

(2) 50 mL of 20 μM lysozyme solution was made from 0.01406 g of lysozyme (MW 14300 Da) in 50 mL of distilled H2O

(3) 30 - 5 mL tubes of 45:1 [Au]:[Lysozme] AuNP solution were prepared with final concentrations of 0.25 mM and 5.56 μM of Au and lysozyme, respectively. Each tubed contained the following:

  • 0.5 mL of 2.5 mM HAuCl4
  • 1.39 mL of 20 μM lysozyme
  • 3.11 mL of distilled H2O

The AuNP solutions were placed in the oven at 80°C for four hours.

2.5 M Tris-HCl/0.5 M CaCl2 pH 8 buffer was prepared by combining 30.285 g of Tris-HCl and 7.351 g of CaCl2 in a 100 mL volumetric flask of DI water. 3 M HCl was added dropwise to a final pH of 8.

0.00055 g of Proteinase K was dissolved in 1 mL of Tris/CaCl2 buffer ---> stock [proteinase K] = 19.03 μM