User:Carly M. Montanero/Notebook/CHEM-571/2013/09/10

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Biomaterials Design Lab: Fall 2013 Main project page
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Objective

The primary way of determining protein concentration is through a measurement of the protein's UV-Vis spectrum and using its molar absorptivity at 280nm to calculate concentration. For low concentrations of proteins, UV-Vis of just the protein is often not sensitive enough to accurately measure concentration. During the semester, we may need to measure protein concentrations that are very low. One chemical tool that we can use to do this is called the Bradford Assay. The Bradford Assay makes use of the Coomassie Blue dye, which binds to proteins. Upon binding to a protein, this dye undergoes a change in its absorption features. (No protein: peak at 460. Protein: peak at around 600). We will be making calibration curves (using the Bradford Assay) for the different proteins we'll be using throughout the semester.

From Dr. Hartings

Procedure

Horseradish Peroxidase Dilution

  1. A stock solution of horseradish peroxidase was created using 10.4 mg of protein in 2 mL of Tris buffer, resulting in a final concentration of 5200 μg/mL.
  2. Six standard samples were created with the following concentrations: 1.9 μg/mL, 2.2 μg/mL, 2.7 μg/mL, 3.0 μg/mL, 3.3 μg/mL, and 7.0 μg/mL.
  • Within each standard sample, the appropriate amount of stock solution was added to an eppendorf tube along with 200 μL of the Bio-Rad Protein Assay reagent. Tris buffer was then added so that the final volume of the standard solutions equaled 1 mL.
3. Each sample was run in the UV-Vis.
4. A blank solution was made with 200 μL of Assay reagent and 800 μL of buffer. This standard was also run in the UV-Vis.
5. This procedure was repeated, creating new standard samples and UV-Vis spectra.

Gold AA/ICPMS Atomic Absorption Standard Preparation

  1. Five diluted solutions of the gold AA/ICPMS stock were made with the following concentrations: 25 μg/mL, 20 μg/mL, 15 μg/mL, 10 μg/mL, and 5 μg/mL.
  • Deionized water was used for the dilution, not the Tris buffer.

Figures

Notes

  • The buffer consisted of 50.02 mM Tris and 50.52 mM NaCl, with a pH of 7.5.
  • The gold stock solution had a concentration of 1000±10 μg/mL.
  • The path length of the cuvette was 0.412 cm.



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