User:Helen L. Slucher/Notebook/CHEM 571/2013/09/10: Difference between revisions

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==Objective==
==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 [http://www.sciencedirect.com/science/article/pii/0003269776905273# Bradford Assay]. The Bradford Assay makes use of the [http://en.wikipedia.org/wiki/Coomassie_Brilliant_Blue Coomassie Blue] dye, which binds to proteins. Upon binding to a protein, this dye undergoes a change in its [http://www.piercenet.com/media/Coomassie-Spectra_350.gif 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.
Make calibration curves, using the Bradford Assay, the protein used.


==Procedure==
==Procedure==

Revision as of 11:15, 12 October 2013

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Objective

Make calibration curves, using the Bradford Assay, the protein used.

Procedure

  1. Make a stock solution that is roughly 10mg protein in 2mL of buffer.
  2. Calculate your actual concentration
  3. 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. Take a UV-Vis (no less than 1 hour after they were produced).
    1. Use the plastic cuvettes.
  5. Make 2 blanks as well (800uL buffer and 200uL Assay reagent) and take it's UV spectrum. (400nm-800nm)
  6. After you have finished one set, repeat the process (make new samples and new measurements)
  7. Make a calibration curve.
  8. Determine if you need to redo any data or sample prep.

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 ug/mL
  2. 20 ug/mL
  3. 15 ug/mL
  4. 10 ug/mL
  5. 5 ug/mL

Data

Making buffer: 1L 50mM Tris 50mM NaCl pH 7.5

Should mass out:

  1. 6.057g Tris
  2. 2.922g NaCl

Actually measured:

  1. 6.0596g Tris
  2. 2.9525g NaCl

Gold solution for group: HAT to make nano particles

  1. Gold solution (HAuCl4·3H2O) 0.0050g in 0.0500mL water → 0.25mM



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