User:Michael S. Bible/Notebook/571/2014/09/10

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Bradford Assay

Procedure taken from Dr. Fox's page

Description

The most direct method for measuring the protein concentration is the use of the Beer - Lambert Law, using published extinction coefficients (molar absorptivities) for the proteins at λ = 280 nm in a UV-VIS spectrum. For low concentrations of proteins, UV-VIS of just the protein is often not sensitive enough to accurately measure concentration. (The limit of detection is about 2 - 3 μM for most proteins.) During the semester, we will likely need to measure protein concentrations that are lower than this. In addition, molar masses and/or extinction coefficients of some proteins are not well quantified. One tool we have can use to measure protein concentrations on the μg/mL level 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. Since this method depends on the number of peptide bonds, concentrations are reported by mass and the method is fairly independent of the particular protein being measured. There are a few interferences, such as co-factors that absorb near λ = 600 nm (e.g. hemes) or basic pH buffers.


Tasklist

The basic protocol can be found here (*Note: use section 2.3, page 5) or here.

  1. Prepare 50 mL of a standard saline solution (0.9 wt-% NaCl). Store in a 45 mL Falcon tube.
  2. Prepare 50 mL of a 50 mM Tris (not Tris-HCl) 50 mM NaCl solution. Store in a 45 mL Falcon tube.
  3. Prepare a stock solution of BSA that is roughly 5 mg in 5 mL of saline.
  4. Calculate your actual solution concentration.
  5. Using a quartz cuvette, record UV-VIS spectra between 200 nm and 800 nm.
    • remember to record UV-VIS spectrum for saline.
  6. Make 6 - 8 standard solutions (1 mL each) between 1 μg/mL and 20 μg/mL. It may be appropriate to use a serial dilution.
    • Determine the appropriate volume of stock solution to use and add it to a 1.5 mL centrifuge tube.
    • Add 200 μL of the Bio-Rad Protein Assay reagent. Use 1:4 concentrate diluted with water.
    • Add the correct amount of Tris/NaCl buffer such that the final volume is 1 mL.
    • Close the tubes and vortex them for 5 - 10 sec.
    • Let them sit for 5 min.
  7. Obtain a UV-VIS spectrum.
    • solutions must be measured within 1 hr of their preparation.
    • use PS cuvettes.
    • record between 400 nm and 800 nm
  8. Make duplicate blanks (4 solutions total) as well
    • 1 mL Tris/NaCl buffer
    • 200 μL Bradford reagent + 800 μL buffer
    • record their UV-VIS spectra between 400 nm and 800 nm
  9. After you have finished, repeat the process using Lysozyme instead of BSA.
  10. Discard solutions in waste bottle and PS cuvettes in the tub or beaker, both in the fume hood.

Data

Image:Bradford_Assay_of_Lysozyme.png

Image:Bradford_Absorbance_v_Concentration.png

Image:Bradford_Absorbance_v_Concentration_1_to_10.png

Image:Bradford_Stock_Solutions.png

Image:Bradford_Stock_Solution_BSA_Corrected_+_purity_calcs.png

Image:Bradford_Corrected_BSA_Absorbance.png

Notes

This area is for any observations or conclusions that you would like to note.


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