1. The term "experimental error" refers to the natural variability inherent in carrying
out any procedure,
i.e. pipetting uncertainties, mixing errors, color development,
and so on. This error relates to inherent assay reproducibility. Estimate the
experimental error, as ± %, for the Bradford assay based upon the known
concentrations of the analyzed proteins. Mention the basis of your estimate. A
given protein may yield different apparent concentrations when analyzed by
different assay procedures. These variations include experimental error plus other
variations due to the nature of the unknown protein and to its behavior in the assay
BCH 367 Elementary Biochemistry Lab
Quantitative Determinations of Proteins Using Spectrophotometry
School of Molecular Sciences, Arizona State University 8
procedure. Based upon what you know about the Bradford assay, offer an
explanation for any error in the determined concentrations.
2. BSA was used as a standard in the Bradford colorimetric assay. For comparison,
use the extinction coefficient at 280 nm for BSA to calculate the concentration of
hemoglobin and lysozyme from their absorbance values at 280 nm (A280).
Compare these values to the actual concentrations of hemoglobin and lysozyme.
What do you conclude from this comparison?
3. Assumed that you have isolated the protein chymotrypsinogen from a cell lysate.
Discuss the advantages and disadvantages of the following protein assays.
a. Absorbance at 215 nm
b. Absorbance at 280 nm
c. The Bradford assay
4. Compare extinction coefficients for DNA (1 mg/ml, 260 nm) and the proteins (1
mg/ml, 280 nm). Explain any difference between the DNA and protein extinction
coefficients in the region of the UV spectrum.
5. Suppose you had a sample that contained equal concentrations of DNA and
protein. Sketch the absorption spectrum you would expect to see in the region
from 240 to 300 nm.
6. BSA has the following profile of amino acid composition compared with the
average of known vertebrate proteins:
AA BSA Average protein
Phenylalanine (Phe) 4.6% 4.0%
Histidine (His) 2.7% 2.9%
Lysine (Lys) 10.1% 7.2%
Arginine (Arg) 3.9% 4.2%
Tryptophan (Trp) 0.3% 1.3%
Tyrosine (Tyr) 3.4% 3.3%
Based upon the given table, is the BSA a good standard to use in the Bradford assay? Explain your answer.
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