User:Beatriz Gimenez De C./Notebook/571/2014/09/10

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Task

  • Prepare 50 mL of a standard saline solution (0.9 wt-% NaCl). Store in a 45 mL Falcon tube.
  • Prepare 50 mL of a 50 mM Tris (not Tris-HCl) 50 mM NaCl solution. Store in a 45 mL Falcon tube.
  • Prepare a stock solution of BSA that is roughly 5 mg in 5 mL of saline.
  • Record UV-VIS spectra between 200 nm and 800 nm.
  • 6 - 8 standard solutions 1 μg/mL and 20 μg/mL. Using determined volume of stock solution to add to 1.5 mL centrifuge tube. Add 200 μL of the Bio-Rad Protein Assay reagent. Use 1:4 concentrate diluted with water. Add to the solution Tris/NaCl until 1 mL volume is reached.
  • vortex tubes and let them sit for 5 min.
  • Obtain a UV-VIS spectrum (between 400 and 800 nm).
  • Make 2 duplicate blanks (1 mL Tris/NaCl buffer and 200 μL BRADFORD reagent + 800 μL buffer)
  • repeat the process using Lysozyme.

Results

  • UV-Vis data from the 6 serial dilutions of BSA (1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL)

Bradfor9-10-14.png

  • UV-Vis data from the 6 serial dilutions of Lysozyme (1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL)

Lysozyme09-10-14.png

Calculations

  • used absorbance values to calculate the concentrations
  • personal note : table yet to be uplaoded ( need to ask how it is done)

Note

  • Worked with Paul because Monika was absent.