User:Daniel-Mario Larco/Notebook/AU Biodesign Lab - 09/03/2013/2013/09/10

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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.

Description

The basic protocol that we will be using for this procedure can be found here. (*Note: use section 2.3, page 5)

  1. Make a stock solution that is roughly 10mg protein in 2mL of buffer. (there are 2-2mL volumetric flasks that you will have to share)
  2. Calculate what your actual concentration
* The concentration of the stock solutions were 5.6μg/mL for the first trial and 5.9μg/mL for the second.
  1. Make 6 standard solutions (1mL each) between 1μg/mL and 10μg/mL
    1. Determine the appropriate volume of your stock to use (for the proper final concentration in 1mL) and add that volume to an eppendorf tube
    2. Add 200μL of the Bio-Rad Protein Assay reagent
    3. Add the correct amount of buffer such that the final volume is 1mL
    4. Close the tubes and vortex them.
    5. Let them sit for 5 minutes
  2. Take a UV-Vis (no less than 1 hour after they were produced).
    1. Use the plastic cuvettes.
  3. Make a blank as well (800uL buffer and 200uL Assay reagent) and take it's UV spectrum.
  4. After you have finished one set, repeat the process (make new samples and new measurements)
  5. Make a calibration curve.
  6. Determine if you need to redo any data or sample prep.
- Because the absorbance of the 2.5μg/mL and 3.0μg/mL were above 1,we made two other samples of 0.25μg/mL and 0.1μg/mL and
reran them and got a new calibration curve.

Here is the calibration curve obtained by our group for Pepsin.

Note - Solutions from today containing the stain will go into a waste container in the hood.

We are also going to make Atomic Absorption standards for tomorrow!

Using the gold AA/ICPMS standard solution make 5 new solutions (Note: Use water - NOT BUFFER - to make these solutions)

  1. 25 μg/mL
  2. 20 μg/mL
  3. 15 μg/mL
  4. 10 μg/mL
  5. 5 μg/mL
 - The standard solutions obtained were put in plastic tubes and saved for the next lab. THey were made by diluting the gold AA/ICPMS standard solution that had a concentration 
of 1000 ± 10 μg/mL. It was diluted with distilled water.


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