User:Douglas M. Fox/Notebook/AU CHEM-571 F2011 Lab Support/2014/09/10

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Today's Experiment - Bradford Assay Objective 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. Prepare 50 mL of a standard saline solution (0.9 wt-% NaCl). Store in a 45 mL Falcon tube. Prepare 50 mL of a 50 mM Tris (not Tris-HCl) 50 mM NaCl solution. Store in a 45 mL Falcon tube. Prepare a stock solution of BSA that is roughly 5 mg in 5 mL of saline. Calculate your actual solution concentration. Using a quartz cuvette, record UV-VIS spectra between 200 nm and 800 nm. remember to record UV-VIS spectrum for saline. 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. 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 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 After you have finished, repeat the process using Lysozyme instead of BSA. Discard solutions in waste bottle and PS cuvettes in the tub or beaker, both in the fume hood. Coordination will be helpful here. Record one spectrum at a time and allow other groups to measure a spectrum in between. Data Analysis First, you will want to find the purity of your protein solution. Using the UV-VIS spectra of your stock solutions, calculate the concentrations of your solutions in both molarity (M or μM) and g/L. The extinction coefficient for BSA is 38,940 M-1cm-1 (λ = 279 nm) and for Lysozyme is 37,800 M-1cm-1 (λ = 280 nm). The purity of the lyophilized powder you used is [UV measured]/[mass measured]. The Bradford reagent has peaks at 460 nm and 630 nm. The Bradford-protein complex has a peak around 600 nm. There will be significant overlap, which will need to be resolved. The best method is to plot a difference spectrum. See an example in my 09/08/2014 notebook entry. Using the peaks of the difference spectra at 600 nm (594 nm in my plot) and the final protein concentration corrected for protein impurities, construct a calibration curve for your two sets of standards. You should find that these curves overlay each other. Determine the minimum concentration (in μM) and maximum concentration (in μM) that you can measure using this analysis for each protein. The minimum occurs at a difference absorbance of 0.05 and the maximum occurs at a difference absorbance of 1.0. You should find that the concentration limits are different despite the same calibration curves. References Many thanks go to Prof. Hartings, who wrote the original protocol for this class.