1. Laboratory 1: Spectrophotometry, Spectroscopy, and Protein Determinations
Objective: To become familiar with the Beer-Lambert Law, A = cl, from determinations of the UV-Vis spectra of
riboflavin and fluorescein; to practice spectroscopic techniques for the quantitative determination of protein
concentrations in solution.
Materials:
Spectrophotometer
Micropipettors
Parafilm
Solutions of riboflavin, fluorescein, 1.0 M TrisCl buffer, pH 8, bovine serum albumin (BSA).
Calculator & a spreadsheet program (e.g., Excel)
Protocol:
Part I. Absorption Spectrum of Riboflavin. At the start of the lab, your TA will provide instructions for powering up
the spectrophotometer. In addition, you will be told how to turn on the deuterium lamp, which is used for readings in the
UV range. (What is the UV range?) Your TA also will give you a solution of known concentration (g/mL) of riboflavin.
From its molecular mass (376 g/mol) and measurements of visible and UV spectra, determine max and the molar
absorption coefficient  (M1 cm1) in the visible region. To obtain these readings you will set your spectrophotometer to
scan from 200 – 600 nm. Dilutions may be necessary to make accurate readings in the spectrophotometer. Remember that
a conservative estimate of the linear range of a spectrophotometer (at least, our spectrophotometers) is 0.1 – 1.0; readings
outside this range generally are not used or are used with caution. If the reading is well above 1.0, dilutions need to be
made until the reading is within the linear range. Ideally, you should repeat the process two more times and report the
average value of the extinction coefficient (or molar absorption coefficient) along with the standard deviation; however,
because of time limitations you will only use the one sample. Remember to clean the cuvettes thoroughly after you are
done. Consider the max found in the visible region. Does this result make sense, given the color of the riboflavin solution?
Comment on this question in the Discussion part of your report.
Part II. Fluorescein. Your TA will give you a solution of fluorescein of unknown concentration and ~0.3 mL of 1.0 M
TrisCl, pH 8 buffer. Use a small test tube to make 2 mL of 0.1 M TrisCl, pH 8 buffer. How are you going to do this?
Hint: remember your old friend: M1V1 = M2V2. Blank the spectrophotometer with 900 L of the 0.1 M TrisCl, pH 8
buffer, then add 100 L of the fluorescein to the cuvette that contains the 900 L and mix thoroughly by tightly covering
the top of the cuvette with Parafilm and inverting several times. (What is the dilution you just made?) Read this sample,
and if the reading is in the appropriate, linear range you can use this reading. If not, adjust your dilution accordingly to get
the sample to read within the linear range. Ideally, you should repeat the process two more times so that you would have
three readings, from which you calculate the average and standard deviation; however, due to time constraints you only
will use one sample that reads within the linear range. Considering the molecular weight of fluorescein (332) and its
extinction coefficient ( 1 / 229
490  mg mL  ), determine the concentration of the original, undiluted solution. (To make this
determination you will have to take into account the “dilution factor.”) Determine also the ratio of its absorption maxima
at 490 nm and 240 nm. From these data, determine the molar absorption coefficient (M1 cm1) of fluorescein at 240 nm.
It is recommended that you again scan from 200 – 600 nm and use the cursors to move to the relevant peaks so that you
can see what the absorbance values are at these peaks.
Part III. Bradford Assay. Your TA will give you a solution of bovine serum albumin (BSA; 1 mg/mL). You will make a
series of appropriate dilutions and plot a standard curve using the following protocol:
1. Prepare a range of standards (1.00 mL volumes in 1.5 mL tubes) containing: 0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, and
0.8 mg/mL of BSA. The fifth sample will be the undiluted (1.0 mg/mL) BSA. You will use these same samples to
construct your standard curve, which is part of the Bradford assay.
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2. 2. Obtain six microcentrifuge tubes (1.5 mL size) and add 990 L of Bradford reagent to each of these tubes. (Your TA
has already diluted the Bradford reagent 1:4.) Next, add 10 L of dH2O to one of the tubes. This tube will be your blank.
Then add 10 L of the 0.2 mg/mL BSA sample to a second tube, 10 L of the 0.4 mg/mL BSA sample to a third tube and
so on until you have used all of the BSA samples. Cap each tube and be sure to label these tubes. Mix the samples by
gentle inversion of the tubes (about 5 times), and allow the samples to stand for 5 minutes. After 5 minutes, add the blank
mixture to one of the cuvettes and zero (or blank) the spectrophotometer at 595 nm. Dispose of the blank solution in the
appropriate waste container and then add the lowest-concentration sample to the same cuvette and read the A595. This
sample can be disposed as described above, and then add the next sample (in order of increasing concentration) to the
same cuvette that held the blank and the first sample. The new sample will be read, disposed of, and the process repeated
until the last, highest-concentration sample is read. When you are done, clean the cuvette you used. You will need to use
an acetone rinse to remove the residual Bradford reagent from this cuvette. Although this method is not ideal (in a perfect
world we would use a clean cuvette for each sample) it should work for our purposes.
Part IV. Standard Curve. Using the data from the solution of BSA (1 mg/mL) that was provided, each student in the
group will plot a standard curve of the absorbance readings at 595 nm (A595) for the Bradford reaction of BSA versus
protein mass (in g). Does your standard curve appear linear? How good is the fit? What statistic allows one to estimate
the “goodness of fit”? Make sure to save this standard curves as you will use it again later in the semester.
Part V. Protein Samples of Unknown Concentration. Your TA will give you different solutions containing different
concentrations of different “unknown” proteins. (We, of course, know the identities and concentrations of these proteins.)
Prepare each of these samples for Bradford analysis as described above and read the A595 of each of these unknowns after
the appropriate incubation period. Did any of your unknown samples absorb outside the linear range? If so, what should
you do to obtain a more accurate estimate of the concentration of this/these particular sample(s)? Make the appropriate
adjustment and proceed until all of your readings are within the linear range of the instrument.
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