User:Melissa Novy/Notebook/CHEM-571/2012/10/03

Objectives

• Use fast protein liquid chromatography (FPLC) on the cell protein solution made on 2012/10/02 to obtain ADA.
• Make 10 mL of Au/BSA solutions at the usual mole ratios (60 through 170 Au:BSA) for atomic absorption. Please refer to the following table for more information.

Obtaining ADA with FPLC

• Binding and elution buffers made on 2012/09/26 were obtained.
• An AKTA Purifier and a 5 mL His nickel column were used to conduct FPLC.

Protocol

• 1. Run about 100 mL of binding buffer through the AKTA system and column at a flow rate of 5 mL/min.
  2. Inject all of the cell protein solution into the system and set the flow rate to 10 mL/min. Note that the composition of the solution is monitored with UV-vis at 280 nm.
  3. After all of the cell protein solution has been run through the 5 mL His nickel column, flush the system with about 200 mL elution buffer at a flow rate of 5 mL/min.
  4. Collect the ADA in elution buffer in glass test tubes and monitor the absorbance with UV-vis to determine the presence of ADA.
  5. Combine the solutions in the test tubes into one Falcon tube and store at 4°C until needed.

Au/BSA Solutions for AA

• Due to a miscalculation, the stock solutions for HAuCl₄ and for BSA were combined into 10 mL of mixed solution in water. The total number of moles of BSA and moles of HAuCl₄ in this new solution were calculated and then new volumes of BSA stock solution were determined:

• There are 0.00000208 mol of HAuCl₄ per 0.00008 L of solution.
• There are 2.4E-10 mol of BSA per 0.00008 L of solution.
• Please refer to the following table for calculations and concentrations:

• The protocol for making the AA Au/BSA solutions is as follows:
  1. Add 80 μL of Au/BSA stock solution to each test tube.
  2. Then add the given volumes of BSA stock solution to each test tube (refer to Volume BSA Added [μL]).
  3. Dilute all solutions to a final volume of 10 mL.
  4. Place the solutions in an 80°C oven for 4 hours.