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

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


==Procedure==
==Procedure==

Revision as of 11:16, 12 October 2013

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Objective

Make calibration curves using the Bradford Assay.

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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