Protein Protocols Protein Protocols

36 Boerner et al.
containing Tris, SDS, urea, and β-mercaptoethanol were treated with nitric acid. As
indicated in Fig. 3B, the presence of increasing amounts of protein resulted in increasing
absorbance at 358 nm, which was again a linear function of protein content. To
confirm that the present assay was useful for measuring polypeptides other than BSA,
we assessed absorbance after reaction of five other polypeptides with nitric acid
including bovine insulin, human fibrinogen, human fibronectin, chicken ovalbumin,
and rat albumin. Each of these polypeptides reacted with nitric acid to produce species
with absorption maxima at 358 nm. In each case, there was a strong correlation (r > 0.99)
between protein content and absorbance (data not shown).
1.2.2. Interfering Substances
Urea, SDS, β-mercaptoethanol, 20% glycerol, and 50% saturated ammonium sulfate
did not interfere with protein determination by the nitric acid method. In addition, the
neutral detergents Tween 20 (1%), Brij 97 (1%), n-octyl α-D-glucopyranoside, and
digitonin (10 µM) did not interfere with this method. In contrast, the presence of 1%
Triton X-100 (a phenol derivative) or trace amounts of phenol produced strong
absorbance at 358 nm that interfered with the assay. Although 1% 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate
(CHAPS) increased absorbance at 358 nm,
it did not interfere with the assay as long as the protein samples used to generate standard
curves were also solubilized in CHAPS.
1.2.3. Comparison of the Nitric Acid Method to Bradford and BCA Protein Assays
To assess the reliability of this technique, one of us blindly measured protein content
in six coded “unknown samples” using the Bradford assay (Coomassie Blue binding),
reaction with BCA, and the nitric acid method. Results of these experiments indicated
that the three methods produce comparable results in assessing protein content in
unknown samples (Fig. 3C). Interestingly, the correlation between actual and measured
(predicted) protein content was slightly higher for the nitric acid method (r = 0.9998)
than for the BCA assay (r = 0.9992) or Bradford assay (r = 0.9991).
1.2.4. Utility of Nitric Acid Method in Loading Extracts of Different Cell Types
for SDS-PAGE
Over the course the past 3 yr, we have had considerable experience in using the
nitric acid method to facilitate the equal loading of SDS-PAGE gels with cell extracts
solublized in electrophoresis sample buffer. Most of our experience has involved using
the method to facilitate equal loading of samples derived from single cell lines on a
given gel (with respect to protein content).
When loading a single gel with samples derived from multiple different cell lines,
however, we noted that the nitric acid method frequently gave the appearance of
unequal loading as assessed by Western blotting for proteins typically used as loading
controls (e.g., histone H1, β-actin). This observation led us to specifically examine
whether the nitric acid method is suited to comparing protein levels between different
cell lines. In assessing this possibility, we ran SDS-PAGE gels with extracts from five
different human cancer cell lines loaded in accord with the amounts of protein in the
extracts indicated by either the BCA or the nitric acid method. When we compared
Western blots for a number of different proteins, we found that both protein assays
facilitated similar gel loading (Fig. 4). Hence, the appearance of unequal gel loading