Saturday, January 30, 2010

Table 1.1. Comparison of various quantitation methodsa


Protein Lowry Bradford BCA Fluorescence
α-Casein 0.91
BSA 1.00 1.00 1.00 1.00
Calf thymus 1.24 1.10 1.15
histones
Chymotryp- 1.52 0.58 0.99
siongen A
Cytochrome c 1.39 1.20 1.11
γ-Globulin 1.07 0.46 0.95
IgG (human) 1.03
IgG (mouse) 1.23 0.79
Lysozyme 1.54 1.00 0.97
Myoglobin 0.84 1.38 0.92 0.91
Ovalbumin 0.93 0.52 1.08 0.97
Ribonuclease A 1.28 0.68
Soybean trypsin 0.83 0.66
inhibitor
Trypsin 1.34 0.34
Mean ± SD 1.18 ± 0.26 0.81 ± 0.34 1.04 ± 0.10 0.96 ± 0.11
a Estimated for highly purified proteins; relative to BSA. Data from:
Peterson GL (1983) Meth Enzymol 91:95; Pierce (1996) Protein assay
technical protocol; and Invitrogen/Molecular Probe Quant-iT Technical
Bulletin (2004)
1.1.1 LOWRY Protein Quantification
1.1.1.1 Standard Procedure
This protocol is slightlymodified,with respect to the original paper
by Lowry et al., to work with smaller volumes. The Folin phenol
method (Lowry protocol; Table 1.2) is useful in the widest variety
of experimental applications and is also the least variable with
different proteins. It is noted that this method, which uses the
oxidation of aromatic amino acids, is easily disturbed by a lot
of substances, which are components of the buffer. As a control
an aliquot of the protein-free buffer in the same volume as the
protein-containing sample has to be taken as blank1.
Since the reaction conditionsmay differ fromexperiment to experiment
and the standard curve is not linear, a couple of standards
with different amounts of protein between 0 and 100 μg should be
measured in each analysis. For most purposes a stock solution of
1 A detailed discussion of Folin–Ciocalteu’s phenol protein determination
method, especially with respect to possible disturbances and
troubles and in comparison with the Bradford method, is given by
Peterson (1996) loc. cit.

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