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

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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:
Stock Solution Concentrations [M] '
[HAuCl4] 0.026
[BSA] 0.000003


  • 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:
Au:BSA Mol BSA Needed Mol BSA Added Volume BSA Added [L] Volume BSA Added [uL]
60 3.46667E-08 3.44267E-08 0.002295111 2295.111111
80 0.000000026 2.576E-08 0.001717333 1717.333333
100 2.08E-08 2.056E-08 0.001370667 1370.666667
120 1.73333E-08 1.70933E-08 0.001139556 1139.555556
128 1.625E-08 1.601E-08 0.001067333 1067.333333
130 0.000000016 1.576E-08 0.001050667 1050.666667
132 1.57576E-08 1.55176E-08 0.001034505 1034.505051
133 1.56391E-08 1.53991E-08 0.001026607 1026.606516
134 1.55224E-08 1.52824E-08 0.001018826 1018.825871
136 1.52941E-08 1.50541E-08 0.001003608 1003.607843
138 1.50725E-08 1.48325E-08 0.000988831 988.8309179
140 1.48571E-08 1.46171E-08 0.000974476 974.4761905
160 0.000000013 1.276E-08 0.000850667 850.6666667
170 1.22353E-08 1.19953E-08 0.000799686 799.6862745


  • 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.