Molecular Biology EU
Exercise 2
Purification and analysis of macromolecules of life
What you need to bring: 1 sheet of graph paper, pencil.
1. Preparation of the standard curve and determination of protein concentration by spectrophotometric measurement at 280 nm.
I. Preparation of standard curve: a. Prepare three solutions of bovine serum albumin (BSA) out of the stock solution (BSA concentration 3mg/ml). Desired concentrations are listed in the
Table 1. Each solution should have the end volume of 100 µl. Calculate how much stock solution and water should be mixed and write it down in Table 1.
Table 1
Desired concentration
µl BSA stock
(3 mg/ml)
1 µg/µl
2 µg/µl
µl H
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0 Absorbance
3 µg/µl b. Solutions should be prepared in Eppendorf test-tubes (max. capacity 1.5 ml).
Each tube should be signed with an appropriate concentration value. c. Turn on Eppendorf BioPhotometer Plus (using on/off button behind the spectrophotometer). Choose “PROTEIN/PROTEIN 280” programme and press
ENTER. d. Prepare blank sample. Transfe r 50 µl of the blank sample to the cuvette. Insert the cuvette into the spectrophotometer pocket. Press BLANK. Remove the cuvette, discard the blank sample, rinse cuvette with distilled water, and dry. e. Transfer 50 µl of the prepared BSA solution to the cuvette. Insert the cuvette into the spectrophotometer pocket. Press SAMPLE. Remove the cuvette, rinse cuvette with distilled water, and dry. Repeat this step for every prepared solutions. f. Write down measured absorbance values for all available wavelengths in
Table 1
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g. Basing on the measured absorbance values, plot standard curve graph
(absorbance (Y-axis) vs concentration (X-axis)).
II. Plotting proteins
’ absorbance spectrum and measuring the protein concentration in the unknown sample (Sample X).
a. Choose “PROTEIN/PROTEIN 280” programme and press ENTER. b. Prepare blank sample. Transfer 50 µl of the blank sample to the cuvette. Insert the cuvette into the spectrophotometer pocket. Press BLANK. Remove the cuvette, discard the blank sample, rinse cuvette with distilled water, and dry. c.
Transfer 50 µl of the given protein sample (Sample X) to the cuvette. Insert the cuvette into the spectrophotometer pocket. Press SAMPLE. Remove the cuvette, rinse cuvette with distilled water, and dry. d. Write down measured absorbance values for all available wavelengths in
Table 2. Write down the concentration measured by the spectrophotometer at
280 nm in Table 3.
Table 2
Wavelength [nm] Absorbance e. Basing on the measured absorbance values, plot protein absorbance spectrum graph (absorbance (Y-axis) vs wavelength (X-axis)). Indicate maximum of the protein absorbance. f. Basing on the standard curve determine the concentration of the unknown protein sample (sample X). Write down the read concentration in Table 3. g. Does the concentration measured by the spectrophotometer and read using standard curve differ? Why?
Table 3
Absorbance at 280 nm
Concentration measured by the spectrophotometer
Concentration determined basing on the standard curve
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